Hyaluronic acid oligosaccharide fractions and drugs containing the same

ABSTRACT

A method for promoting expression of a heat shock protein, or for inhibiting cell injury or cell death, or for treating a disease for which cell or tissue protection is desired, or for promoting production of IL-10, or for inhibiting production of IL-8, by administering an effective amount of a fraction containing a hyaluronic acid tetrasaccharide comprising four saccharide residues and having certain physicochemical properties or by administering an effective amount of an isolated and substantially pure tetrasaccharide of formula (1) described in the application. Methods for preserving an organ.

This application is a Continuation application of U.S. Ser. No.11/622,673, filed Jan. 12, 2007, now pending; which is a Divisionalapplication of U.S. application Ser. No. 10/312,879, filed Jun. 30,2003, now patented; which is a National Stage application ofPCT/JP01/05918, filed Jul. 26, 2001; and claims priority to JapaneseApplications 2000-206404, filed Jul. 7, 2000; and 2000-247840, filedAug. 17, 2000.

TECHNICAL FIELD

The present invention relates to hyaluronic acid oligosaccharides(henceforth also referred to as “HA oligosaccharides”) and novelfractions thereof.

The present invention also relates to drugs containing the HAoligosaccharides (in particular, a heat shock protein expressionpromoter, cell death inhibitor, cell injury inhibitor or cell and tissueprotecting agent (especially those used for applications as an organpreservation agent, antiulcer agent and antihepatopathic agent, IL-10production promoter or IL-8 production inhibitor)) as activeingredients.

BACKGROUND ART

It is known that activities and functions of hyaluronic acid (henceforthalso referred to as “HA”) change depending on the molecular size. Forexample, it has been reported that HA having a molecular weight of1,200,000 shows NF-κB inactivation activity, neovascularizationinhibition activity and so forth (Neumann A., Schinzel R., Palm D.,Riederer P. and Munch G., “High molecular weight hyaluronic acidinhibits advanced glycation endproduct-induced NF-κB activation andcytokine expression”, FEBS Lett., 453(3):283-7, Jun. 25, 1999; FeinbergR. N., Beebe D. C., Hyaluronate in vasculogenesis, Science220:1177-1179, 1983), whereas HA having a molecular weight of 500,000 orless has inverse activities (Noble P. W., McKee C. M., Cowman M. andShin H. S., “Hyaluronan fragments activate an NF-κB/I-κB alphaautoregulatory loop in murine macrophages”, J. Exp. Med. 183:2373-2378,1996; West D. C. and Shaw D. M., “Tumour hyaluronan in relation toangiogenesis and metastasis”, In: Laurent T. C. ed., The chemistry,biology and medical applications of hyaluronan and its derivatives,London: Portland Press, 1998:227).

It can be said that this well suggests possibilities of finding ofvarious activities also for HA oligosaccharides and finding of specificactivities depending on sizes of HA oligosaccharides. Therefore, it canbe considered that, if HA oligosaccharides having different sizes arecombined, they may exert an additive or synergistic effect, orconversely, against an activity of an HA oligosaccharide of a certainsize, another HA oligosaccharide having a different size may act as anantagonist.

Assuming as described above, if a fraction containing HAoligosaccharides of various sizes is used for search of activitiesthereof for development of drugs utilizing HA oligosaccharides, not onlyit cannot be found which size of HA oligosaccharide constitutes anentity of a certain activity, but also an activity of HA oligosaccharideof a certain size may be compensated by an HA oligosaccharide of anothersize. Thus, important physiological activities or functions hidden in HAoligosaccharides may be overlooked.

Further, when an HA oligosaccharide of a certain specific size is usedas a drug, it is necessary to eliminate oligosaccharides of other sizesthat inhibit the function exerted by the HA oligosaccharide of specificsize as much as possible, and there has been desired a fraction of highpurity that does not substantially contain substances undesirable fordrugs.

As described above, for creation and provision of novel drugs, there hasbeen desired a HA oligosaccharide fraction of high purity that consistsof HA oligosaccharides of substantially uniform sizes and does notsubstantially contain HA oligosaccharides of other sizes and otherimpurities.

Meanwhile, heat shock proteins (henceforth also referred to as “Hsp”)are also called stress proteins, which are proteins that inhibitaffections caused by various stress reactions, and various familiesthereof have been known. The heat shock proteins of Hsp70 family, whichis one of the families, are considered to prevent cell injury or celldeath through actions of inhibiting structural changes of proteins,production of abnormal proteins and so forth caused by factorsgenerating environmental stresses such as heat shock, hydrogen peroxide,heavy metals, amino acid analogues and glucose depletion and stressfactors such as fervescence, inflammation, ischemia, viral infection,metabolic disorders, hypercardia, oxidative stresses, cellular tissueaffections, oncogenes and carcinogenic substances or actions ofregenerating functions of proteins.

In connection with the above, a stress protein expression promotercontaining HA as an active ingredient is disclosed in Japanese PatentUnexamined Publication (Kokai) No. 9-227386. In this patent document, itis described that HA consisting of about 2 to 20 saccharides ispreferred as the active ingredient (page 3), and it was suggested thatHA consisting of ten or less saccharides was involved in enhancement orinduction of expression of stress proteins (page 6). There is alsodescribed an experiment demonstrating that an unsaturated HAdisaccharide enhanced expression of a stress protein and inhibited celldeath (Example 2).

However, the above patent document does not specifically disclosevarious oligosaccharides as in the present invention, and it does notteach nor suggest at all the extremely notable activity for enhancementof expression of stress protein specifically observed for HAtetrasaccharide mentioned later.

Further, considering the functions of the aforementioned stressproteins, the stress proteins are considered to be involved also inprotection of cells and tissues.

Concerning the above, Japanese Patent Unexamined Publication No.11-246301 discloses an organ preservation solution for use in organtransplantation containing HA and/or a physiologically acceptable saltthereof. Although this patent document describes that the averagemolecular weight of HA is preferably 100,000 or more, it does notdescribe nor suggest HA oligosaccharides and superior effects exerted bythem.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a fraction thatcontains HA oligosaccharides having a size of about 4 to 60 saccharides,in particular, HA oligosaccharide of a substantially uniform size, anddoes not substantially contain HA oligosaccharides of other sizes andother impurities.

Another object of the present invention is to provide a useful drugcontaining such HA oligosaccharides as an active ingredient, especiallya more effective Hsp expression promoter, cell death inhibitor or cellinjury inhibitor. A further object of the present invention is toprovide a cell and tissue protecting agent and provide an organpreservation agent, antiulcer agent and antihepatopathic agent, IL-10production promoter and IL-8 production inhibitor utilizing it.

The inventors of the present invention assiduously studied in order toachieve the aforementioned objects. As a result, they obtained novelfractions of HA oligosaccharides that consisted substantially only ofoligosaccharide of a desired size and did not substantially containoligosaccharides of other size and other impurities by fractionating HAoligosaccharides having a size selected from sizes of 4 to 60saccharides and a mixture of such oligosaccharides with a particularmethod.

Thus, the present invention provides an HA oligosaccharide having a sizeselected from sizes of 4 to 60 saccharides (henceforth also referred toas “oligosaccharide of the present invention”).

The present invention also provides a fraction containing theoligosaccharide of the present invention and having the physicochemicalproperties defined in the following (1) to (6) (henceforth also referredto as “fraction of the present invention”).

(1) If the fraction is analyzed by the detection methods mentioned inthe following (a) and (b) using gel filtration chromatography, thefraction shows a substantially single peak in both of the methods, andthe peaks have peak areas defined in the following (a) and (b).(a) If the detection is performed based on absorbance at 210 nm, arelative ratio of peak area of the substantially single peak to the sumof peak areas of total HA oligosaccharides in the fraction is 85% ormore.(b) If the detection is performed by using a differential refractometer,a relative ratio of peak area of the substantially single peak to thesum of peak areas of total HA oligosaccharides in the fraction is 98% ormore.(2) If the fraction is analyzed based on absorbance at 210 nm by usinganion exchange chromatography, the fraction shows a substantially singlepeak, and a relative ratio of peak area of the substantially single peakto the sum of peak areas of total HA oligosaccharides in the fraction is90% or more.(3) If oligosaccharides in the fraction are labeled with fluorescenceand then analyzed by electrophoresis, a single band is detected, andbands of HA oligosaccharides of other sizes are not detected.(4) If a theoretical value of monoisotopic molecular weight or averagemolecular weight of HA oligosaccharides constituting the fraction istaken as 1, an actual value of the same measured for the fraction bymass spectrometry is 0.997 to 1.003 (relative value).(5) Respective differences between theoretical values (weight %) ofcarbon (C), hydrogen (H) and nitrogen (N) contents in HAoligosaccharides constituting the fraction and values actually measuredfor the elements by elemental analysis of the fraction (weight %) areall in the range of ±1 (weight %).(6) The fraction does not substantially contain proteins, DNA andendotoxins.

The fraction of the present invention preferably further shows thephysicochemical property defined in the following (7).

(7) Results of ¹H-NMR and ¹³C-NMR of the fraction do not contradict tothe structure of HA oligosaccharide represented by the following formula(1).

(In the formula, n is an integer of 1 to 29, M represents a proton or amonovalent cation, and Ac represents acetyl group.)

The size of HA oligosaccharides contained in the fraction of the presentinvention is preferably selected from sizes of 4 to 20 saccharides, morepreferably selected from sizes of 4 to 16 saccharides, still morepreferably selected from sizes of 4 to 14 saccharides, and it is mostpreferably a size of 4 saccharides (tetrasaccharides).

Moreover, the inventors of the present invention investigatedpossibility of use of HA oligosaccharides of the present invention as adrug. As a result, they found that an HA oligosaccharide of a particularsize had extremely notable Hsp expression promoting action, cell deathinhibitory action and cell injury inhibitory action, and therebyprovided an Hsp expression promoter, cell death inhibitor and cellinjury inhibitor that can achieve the aforementioned object.

The inventors of the present invention further found that theoligosaccharide of the present invention had a superior cell and tissueprotection action, and thus further provided a cell and tissueprotecting agent as well as an organ preservation agent, antiulceragent, antihepatopathic agent, IL-10 production promoter and IL-8production inhibitor utilizing it.

That is, the present invention provides a drug (pharmaceuticalcomposition, also referred to as “drug of the present invention”hereinafter) containing the oligosaccharide of the present invention asan active ingredient. This drug is preferably an Hsp expressionpromoter, cell death inhibitor, cell injury inhibitor or cell and tissueprotecting agent.

Moreover, the cell and tissue protecting agent is preferably used forapplications as an organ preservation agent, antiulcer agent,antihepatopathic agent, IL-10 production promoter and IL-8 productioninhibitor.

The drug and agents of the present invention preferably contain, as anactive ingredient, an HA oligosaccharide having a size selected fromsizes of 4 to 20 saccharides, more preferably an HA oligosaccharidehaving a size selected from sizes of 4 to 16 saccharides, still morepreferably an HA oligosaccharide having a size selected from sizes of 4to 14 saccharides, most preferably an HA oligosaccharide having a sizeof 4 saccharides.

As described above, physiological activities and functions of HAoligosaccharides had also hitherto been reported. However, there havehitherto been no examples of use of HA oligosaccharides guaranteed onmany aspects such as molecular size, molecular structure and purity(contents of oligosaccharides of other sizes and contents of othercomponents such as proteins and DNA, contents of endotoxins). That is,as for the physiological activities and functions of HA oligosaccharidesmentioned in the previous reports, influences of contaminants could notbe denied. On the other hand, in the present invention, a fraction of HAoligosaccharide not substantially containing other components such asproteins and DNA or such an HA oligosaccharide not substantiallycontaining HA oligosaccharides of other sizes was obtained, andphysiological activities and functions thereof were elucidated.

Hereafter, the present invention will be explained in detail.

<1> Oligosaccharide of the Present Invention

The oligosaccharide of the present invention is an HA oligosaccharidehaving a size selected from sizes of 4 to 60 saccharides.

The “HA oligosaccharide” used herein is an oligosaccharide having acomposition similar to the constitutive disaccharide composition of HA.Specifically, it refers to an oligosaccharide consisting of alternatelylinked residues of glucuronic acid (GlcA) and N-acetylglucosamine(GlcNAc), which are constitutive monosaccharides of HA.

That is, the HA oligosaccharide include an oligosaccharide havingglucuronic acid residue at the non-reducing end, of which typicalexample is the structure represented by the aforementioned formula (1),as well as an oligosaccharide having N-acetylglucosamine residue at thenon-reducing end.

The saccharide locating at the non-reducing end may be a saturatedsaccharide (monosaccharide not containing a double bond as acarbon-carbon bond) or an unsaturated saccharide (monosaccharidecontaining a double bond as a carbon-carbon bond). In the followingexplanations, GlcA represents saturated glucuronic acid residue andΔGlcA represents unsaturated glucuronic acid residue.

An oligosaccharide whose saccharide locating at the non-reducing end isa saturated saccharide is especially preferred. Specifically, HAoligosaccharides represented by the following formula (2) are preferred.

GlcA(-GlcNAc-GlcA)n-GlcNAc  (2)

(In the formula, GlcA represents a glucuronic acid residue, GlcNAcrepresents an N-acetylglucosamine residue, — represents a glycosidiclinkage, and n represents an integer of 1 to 29.)

The glycosidic linkage of GlcA-GlcNAc in the aforementioned formula (2)is preferably a β1->3 linkage, and the glycosidic linkage in GlcNAc-GlcAis preferably a β1->4 linkage.

Among those, HA oligosaccharides represented by the following formula(1) are particularly preferred.

(In the formula, n is an integer of 1 to 29, M represents a proton or amonovalent cation, and Ac represents acetyl group.)

However, those having an unsaturated saccharide as a saccharide locatingat the non-reducing end also have significant physiological activity asdemonstrated in the examples mentioned later, and they are alsopreferred embodiments of the present invention. Specific examplesthereof include an HA oligosaccharide represented by the followingformula.

(In the Formula, Ac Represents an Acetyl Group.)

An HA oligosaccharide represented by the aforementioned formula andhaving an unsaturated saccharide at the non-reducing end corresponds toΔHA4 used in the examples mentioned later.

Further, the oligosaccharide of the present invention may be in the formof a salt, and may be in an ionized state. Examples of the salt include,for example, salts with an inorganic base such as alkali metal salts(sodium salt, lithium salt, potassium salt etc.), alkaline earth metalsalts and ammonium salts and salts with an organic base such asdiethanolamine salts, cyclohexylamine salts, amino acid salts,galactosamine salts and glucosamine salts. Among these, alkali metalsalts are preferred, and sodium salts are particularly preferred.

A source of the oligosaccharide of the present invention is notparticularly limited. For example, the oligosaccharide of the presentinvention may be produced by a process comprising separation andpurification of HA from chicken crest, umbilical cord, porcine skin,bovine skin, skins or aortas of fish and other animals, microorganismsproducing HA and so forth and degradation of HA (e.g., enzymaticdegradation, chemical degradation, heat treatment, ultrasonicationetc.). The oligosaccharide of the present invention may also be producedby a synthetic process (e.g., chemical synthesis and enzymaticsynthesis).

Examples of the enzymatic degradation method include methods of allowingan enzyme that degrades HA such as hyaluronidase (derived from testis),hyaluronidase (derived from Streptomyces), hyaluronidase SD,chondroitinase ACI, chondroitinase ACIII, chondroitinase ABC andendoglucuronidase (derived from leech) to act on HA (refer to ShinSeikagaku Jikken Koza [Lecture of Biochemical Experiments, New Edition],“Saccharide II, Proteoglycan and Glycosaminoglycan”, published by TokyoKagaku Dojin, pp. 244-248, 1991). In order to obtain an HAoligosaccharide of the aforementioned formula (1), a hydrolase ispreferably used as the enzyme that degrades HA.

Examples of the chemical degradation method include the alkalinedecomposition method, dimethyl sulfoxide method (DMSO method) and soforth. The alkaline decomposition method can be specifically performedby, for example, adding a base such as about 1 N sodium hydroxide to asolution of HA, warming the mixture for several hours to degrade HA intothose of lower molecular weights and then neutralizing the mixture withaddition of an acid such as hydrochloric acid. Examples of the DMSOmethod include the method of Nagasawa et al. (Carbohyd. Res., 141, pp.99-110, 1985). The hydrolysis can also be carried out by using an acidsuch as hydrochloric acid and sulfuric acid.

Examples of the ultrasonication method include the method described inBiochem., 33, pp. 6503-6507, 1994 and so forth.

Examples of the synthesis method include the methods described inGlycoconjugate J., pp. 453-439, 1993; International Patent PublicationWO93/20827 and so forth.

The oligosaccharide of the present invention produced as described abovecan be purified to a desired purity. It can be purified to such a degreethat the oligosaccharide of the present invention should substantiallyconsist of oligosaccharide of a uniform size and not substantiallycontain HA oligosaccharides of other sizes and other impurities, forexample, as described below.

<2> Fraction of the Present Invention

The fraction of the present invention is a fraction containing theoligosaccharide of the present invention and showing the specificphysicochemical properties described later.

Although the oligosaccharide of the present invention (fractioncontaining oligosaccharide of the present invention, different from thefraction of the present invention) by the method described in the abovesection <1>, the obtained fraction contains HA oligosaccharides ofmultiple kinds of sizes as an admixture. On the other hand, the fractionof the present invention comprises HA oligosaccharide of a substantiallyuniform size, and does not substantially contain HA oligosaccharides ofother sizes and other impurities. The fraction of the present inventioncan be produced by the method described below.

The fraction obtained by the method described in the above <1> (fractioncontaining oligosaccharides of various sizes as an admixture) is appliedto a column of strongly basic anion exchanger. Examples of the stronglybasic anion exchanger include anion exchangers having trimethylammoniumgroups, trimethylammoniomethyl groups, β-hydroxyethyldimethylammoniumgroups, 2-hydroxypropylamino groups, diethyl-(2-hydroxypropyl)aminoethylgroups, dimethylethanolammonium groups or the like, and an anionexchanger having trimethylammoniomethyl groups is preferred.

Although size of the column can be suitably selected depending on theamount to be applied and so forth, a column diameter of 1 to 5 cm and acolumn length of about 50 to 150 cm are preferred for the preparation ina small scale. Further, two or more of such columns are preferablycombined.

If a fraction containing HA oligosaccharides of multiple kinds of sizesas an admixture is applied to such a column, HA oligosaccharidescontained in the fraction bind to the strongly basic anion exchanger inthe column via ionic bonds.

The bound HA oligosaccharides can be eluted with a salt concentrationgradient. The salt used for the elution is not particularly limited, andNaCl, KCl, LiCl etc. can be used. However, NaCl is preferably used.

The salt concentration gradient is preferably started at a saltconcentration of about 0 to 0.1 M and increased to about 0.5 M. Further,the salt concentration gradient is preferably linear, and the saltconcentration is preferably increased with a rate of about 0.1 M/40 to50 hours, more preferably about 0.1 M/45 to 50 hours.

Further, the flow rate is preferably 80 to 100 mL/hour, more preferably85 to 95 mL/hour.

By selecting such chromatographic conditions, HA oligosaccharides boundto the strongly basic anion exchanger are eluted in the order of sizestarting with the oligosaccharide of the smallest size. Based on this,it is considered that the binding power of the HA oligosaccharides tothe strongly basic anion exchanger is substantially proportional to thesize of HA oligosaccharides, and as a result, it is considered that theoligosaccharides are eluted in the order of binding power starting withthe oligosaccharide of the smallest power (the oligosaccharide of thesmallest size). The fraction of the present invention is provided forthe first time by utilizing such binding and elution characteristics ofthe strongly basic anion exchanger and HA oligosaccharides. According tothis method, HA oligosaccharides of various sizes can be separated in asimple manner by one column operation with high resolution toefficiently produce a HA oligosaccharide fraction of high puritycomprising HA oligosaccharide of a substantially uniform size and notsubstantially containing HA oligosaccharides of other sizes and othersimpurities (fraction of the present invention).

The HA oligosaccharides contained in the fraction of the presentinvention preferably has a size selected from sizes of 4 to 20saccharides, more preferably selected from sizes of 4 to 16 saccharides,still more preferably selected from sizes of 4 to 14 saccharides, and itis most preferably a size of 4 saccharides.

The fraction of the present invention obtained as described above may besubjected again to the aforementioned chromatography step. Further, itmay also be subjected to concentration, desalting and other treatments.

The form of the fraction of the present invention for storage,distribution etc. is not particularly limited, and it may be in the formof solution, frozen product, lyophilized product or the like.

Further, the HA oligosaccharides in the fraction of the presentinvention may be in the form of salt, and may be in an ionized state.Examples of the salt include, for example, salts with an inorganic basesuch as alkali metal salts (sodium salt, lithium salt, potassium saltetc.), alkaline earth metal salts and ammonium salts and salts with anorganic base such as diethanolamine salts, cyclohexylamine salts, aminoacid salts, galactosamine salts and glucosamine salts. Among these,alkali metal salts are preferred, and sodium salts are particularlypreferred. When the fraction of the present invention is used for thedrug of the present invention explained below, pharmaceuticallyacceptable salts among these salts can be used. Also in this case,sodium salts are particularly preferred.

The fraction of the present invention obtained as described above ischaracterized by satisfying all the physicochemical properties definedin the following (1) to (6). The numeric values and so forth mentionedin the physicochemical properties mentioned below may somewhat changewith conditions of tests or experiments, environment, equipments used,degree of skill of laboratory worker, other factors and so forth.Therefore, the numeric values must not be construed to be strictlylimited to the numeric values themselves, and should be construed inconsideration of existence of small differences in measurement resultsdue to experimental conditions etc. (differences of numeric valuesacceptable from a commonsense standpoint of those skilled in the art,differences of numeric values in such a degree that they can berecognized as those of the same fraction by those skilled in the art).

(1) If the fraction is analyzed by the detection methods mentioned inthe following (a) and (b) using gel filtration chromatography, thefraction shows a substantially single peak in both of the methods, andthe peaks have peak areas defined in the following (a) and (b)(a) If the detection is performed based on absorbance at 210 nm, arelative ratio of peak area of the substantially single peak to the sumof peak areas of total HA oligosaccharides in the fraction is 85% ormore. This relative ratio of peak area is preferably 90% or more, morepreferably 95% or more.(b) If the detection is performed by using a differential refractometer(also abbreviated as “RI” hereinafter), a relative ratio of peak area ofthe substantially single peak to the sum of peak areas of total HAoligosaccharides in the fraction is 98% or more.

This physicochemical property indicates that the fraction is constitutedby HA oligosaccharide molecules of a substantially uniform size. It alsosuggests that the fraction does not substantially contain otherimpurities.

In this specification, the expression of “substantially single(substantially uniform)” does not mean “completely single (completelyuniform)”, but means that “recognized to be single (uniform) by thoseskilled in the art”. For example, it is generally difficult to obtain afraction of oligosaccharide of a large size not containingoligosaccharides of other sizes at all, and this is a common sense inthis technical field. Therefore, in chromatography analysis, massspectrometry analysis or the like of a fraction of oligosaccharide of alarge size, even if, in addition to a major peak of the concernedoligosaccharide, a small peak of oligosaccharide of another size isobserved, it is usually recognized as a substantially single peak bytaking the sizes of oligosaccharides into consideration. Therefore, insuch a case, it can be considered “substantially single peak”.

(2) If the fraction is analyzed based on absorbance at 210 nm by usinganion exchange chromatography, the fraction shows a substantially singlepeak, and a relative ratio of peak area of the substantially single peakto the sum of peak areas of total HA oligosaccharides in the fraction is90% or more. This relative ratio of peak area is preferably 95% or more.

This physicochemical property indicates that the fraction is constitutedby HA oligosaccharide molecules having substantially uniform valence ofcharge, i.e., homogenous HA oligosaccharides. It also suggests that thefraction does not substantially contain other impurities.

The HA oligosaccharide molecules regularly contain carboxyl groups, forexample, as shown in the formula (1), and these groups are ionized andconverted into —COO⁻ ions in a solution. Therefore, it can be said thatthe number of the —COO⁻ ions contained in HA oligosaccharide moleculesof a constant size is constant. Therefore, a substantially uniformvalence of charge of HA oligosaccharides constituting the fraction alsoindicate that the fraction is constituted by HA oligosaccharide of asubstantially uniform size.

(3) If oligosaccharides in the fraction are labeled with fluorescenceand then analyzed by electrophoresis, a single band is detected, andbands of HA oligosaccharides of other sizes are not detected.

This indicates that it is also clarified that the fraction isconstituted by HA oligosaccharide molecules of a substantially uniformsize by analysis using a technique other than chromatography(electrophoresis), and supports the result of the chromatographicanalysis. It also suggests that the fraction does not substantiallycontain other impurities.

(4) If a theoretical value of monoisotopic molecular weight or averagemolecular weight of HA oligosaccharides constituting the fraction istaken as 1, an actual value of the same measured for the fraction bymass spectrometry is 0.997 to 1.003 (relative value). This relativevalue is preferably in the range of 0.998 to 1.002, more preferably inthe range of 0.999 to 1.001.

This also suggests that HA oligosaccharides constituting the fractionsubstantially consist of a single kind of oligosaccharides as also forthe molecular weight (mass) and do not substantially contain otherimpurities.

(5) Respective differences between theoretical values (weight %) ofcarbon (C), hydrogen (H) and nitrogen (N) contents in HAoligosaccharides constituting the fraction and values of the sameactually measured for the elements by elemental analysis of the fraction(weight %) are all in the range of ±1 (weight %).

If the HA oligosaccharides are in the form of sodium salt, it ispreferred that the difference for sodium (Na) should be similarly in therange of ±1 (weight %)

This suggests that the HA oligosaccharides constituting the fraction issubstantially uniform as also for the elemental composition and does notsubstantially contain other impurities.

(6) The fraction does not substantially contain proteins, DNA andendotoxins.

Although content of proteins in the fraction of the present inventioncan be measured by using a conventional method well known to thoseskilled in the art, the Lowry method is preferred. When proteins cannotbe detected by this method (when the content is below the detectionlimit), it is determined that proteins are not substantially contained.

Although content of DNA in the fraction of the present invention can bemeasured by using a conventional method well known to those skilled inthe art, the threshold method is preferred. When DNA cannot be detectedby this method (when the content is below the detection limit), it isdetermined that DNA is not substantially contained.

Although content of endotoxins in the fraction of the present inventioncan be measured by using a conventional method well known to thoseskilled in the art, the Limulus test method using horseshoe crabamebocyte lysate is preferred. When endotoxins cannot be detected bythis method (when the content is below the detection limit), it isdetermined that endotoxins are not substantially contained.

In the present specification, the expression of “do not substantiallycontain” does not mean “do not contain even one molecule” of impurity orthe like, but means such a degree of content that those skilled in theart should recognize that impurities etc. are not contained (such adegree of content that impurities etc. cannot be detected by the methodsmentioned above). If detection at a molecular level becomes possible infuture thanks to progress of analytical techniques, the expression of“do not substantially contain” must be interpreted based on thedescriptions of the present application.

Further, in one of preferred embodiments of the fraction of the presentinvention, HA oligosaccharide of a substantially uniform size havesaturated glucuronic acid residues at the non-reducing ends, and in sucha case, the fraction of the present invention has the property definedin the following (7).

(7) Results of ¹H-NMR and ¹³C-NMR of the fraction do not contradict tothe structure of HA oligosaccharide represented by the following formula(1)

(In the formula, n is an integer of 1 to 29, M represents a proton or amonovalent cation, and Ac represents acetyl group.)

This indicates that the HA oligosaccharides constituting the fractionare HA oligosaccharides represented by the aforementioned formula, andalso suggests that the HA oligosaccharides are substantially uniform asalso for the molecular structure and the fraction does not substantiallycontain other impurities.

<3> Drug of the Present Invention

The drug of the present invention is a drug containing theoligosaccharide of the present invention as an active ingredient.

The oligosaccharide of the present invention used as an activeingredient of the drug of the present invention, preferred embodimentsthereof and so forth are as explained in the above <1>.

In particular, preferably used as the oligosaccharide of the presentinvention is an oligosaccharide having a size selected from sizes of 4to 20 saccharides (HA tetrasaccharide to HA icosasaccharide), morepreferably used is an oligosaccharide represented by the formula (2)wherein n is selected from integers of 1 to 9, and particularlypreferably used is an oligosaccharide represented by the formula (1)wherein n is selected from integers of 1 to 9.

More preferably, an oligosaccharide of the present invention having asize selected from sizes of 4 to 16 saccharides (HA tetrasaccharide toHA hexadecasaccharide) is preferably used, an oligosacchariderepresented by the formula (2) wherein n is selected from integers of 1to 7 is more preferably used, and an oligosaccharide represented by theformula (1) wherein n is selected from integers of 1 to 7 isparticularly preferably used.

Still more preferably, an oligosaccharide of the present inventionhaving a size selected from sizes of 4 to 14 saccharides (HAtetrasaccharide to HA tetradecasaccharide) is preferably used, anoligosaccharide represented by the formula (2) wherein n is selectedfrom integers of 1 to 6 is more preferably used, and an oligosacchariderepresented by the formula (1) wherein n is selected from integers of 1to 6 is particularly preferably used.

Among these, preferably used as the oligosaccharide of the presentinvention is an oligosaccharide of a size of 4 saccharides (HAtetrasaccharide), more preferably used is an oligosaccharide representedby the formula (2) wherein n is 1, and particularly preferably used isan oligosaccharide represented by the formula (1) wherein n is 1. Byusing HA tetrasaccharide, various superior pharmacological actions canbe exerted.

Although the aforementioned formulas (1) and (2) represent anoligosaccharide of the present invention having a saturated glucuronicacid residue at the non-reducing end, an oligosaccharide of the presentinvention having an unsaturated glucuronic acid residue at thenon-reducing end may be used for the drug of the present inventiondepending on the specific use of the drug of the present invention, andthe preferred size thereof is as described above.

More preferably, the fraction of the present invention is used as anactive ingredient of the drug of the present invention. The fraction ofthe present invention and preferred embodiments thereof used for thepreparation of the drug are as described in the above section <2>.Further, the preferred sizes of HA oligosaccharides contained in thefraction of the present invention are the same as described above.

By using the fraction of the present invention, content of HAoligosaccharides in the drug of the present invention can be increased,and the drug of the present invention that does not substantiallycontain a substance of which contamination is not allowed as a drug canbe produced.

The drug of the present invention can be prepared by a known method.Upon preparation of the drug, components usually used for drugs such asother pharmaceutically active ingredients, conventional stabilizers,emulsifiers, osmotic pressure regulators, pH regulators, buffers,isotonic agents, preservatives, soothing agents, colorants, excipients,binders, lubricants, disintegrating agents and so forth can be used, solong as such components do not adversely affect the HA oligosaccharides,which are the active ingredient of the drug of the present invention,and do not affect efficacy of the drug of the present invention.

In addition, although the drug of the present invention preferablycontains the fraction of the present invention as an active ingredient,it is sufficient that the drug should contain at least an HAoligosaccharide having a size selected from sizes of 4 to 60saccharides, since it contains such an HA oligosaccharide as an activeingredient, and the drug may contain an HA oligosaccharide of anothermolecular size or HA having a large size that cannot be referred to asoligosaccharide so long as it does not affect the efficacy of the drugof the present invention.

The drug of present invention is preferably a drug for use in promotionof Hsp expression, inhibition of cell death, inhibition of cell injuryor protection of cell and tissue, and it can be any one of the followingagents depending on the use.

[1] Hsp Expression Promoter

The term “promotion of expression” used in the present specificationincludes the meanings of both of “increasing of expression amount” and“enhancement of activity”. Therefore, the term “heat shock proteinexpression promoter (Hsp expression promoter)” used in the presentspecification includes both of “an agent for increasing expressionamount of heat shock protein (Hsp)” and “an agent for enhancing activityof Hsp”.

(1) Administration Method, Dosage Form Etc. of Hsp Expression Promoter

When the drug of the present inventions is used as an Hsp expressionpromoter, an HA oligosaccharide consisting of 4 saccharides among HAoligosaccharides is particularly preferably used. Such an HAoligosaccharide exerts extremely superior Hsp expression promotingactivity.

Although the administration method for the Hsp expression promoter isnot particularly limited so long as the Hsp expression promoting actionof HA oligosaccharide is exerted, examples thereof include, for example,injection (intravenous, intramuscular, subcutaneous, intracutaneous,intraperitoneal injections etc.), nasal administration, oraladministration, transdermal administration, inhalation and so forth. Anappropriate dosage form can be selected depending on the administrationmethod, and there can be employed injections (solutions, suspensions,emulsions, solid agents for dissolution upon use etc.), tablets,capsules, solutions, granules, powders, liposomes, ointments, plasters,lotions, dermatologic pastes, patches, gels, suppositories, powders forexternal application, sprays, inhalation powders and so forth.

Although the content of HA oligosaccharide (especially HAtetrasaccharide) in the Hsp expression promoter is not also particularlylimited, it is preferably 0.1 to 10% (w/v), for example, when the Hspexpression promoter is provided as an injection.

When the Hsp expression promoter is provided as an injection, forexample, it may be in the form of any of solution, frozen preparationand lyophilized preparation. These may be filled and sealed in anappropriate container such as ampoule, vial and syringe for injection,distributed or stored as they are, and administered as an injection.

Although HA tetrasaccharide is particularly preferred as an activeingredient of the Hsp expression promoter as described above, HAoligosaccharides of other molecular sizes may be further contained.However, as is demonstrated by the examples described later, HAtetrasaccharide specifically exhibits particularly marked Hsp expressionpromoting action. Therefore, if the content of HA tetrasaccharide isincreased, correspondingly higher effect can be obtained, and the dosemay be decreased while the effect is maintained at the same level as HAsaccharide of the other sizes. Thus, it is particularly preferred thatthe HA oligosaccharides contained in the Hsp expression promoter shouldsubstantially consist only of the tetrasaccharide, and it is desirablethat HA oligosaccharides other than HA tetrasaccharide should not beadded.

(2) Objectives of Administration of Hsp Expression Promoter Etc.

The Hsp expression promoter is expected to be effective against manydiseases caused by cell injury or cell death for which preventive effectby Hsp is suggested, for example, ischemic diseases caused byangiostenoses or ischemia such as heart diseases (myocardial infarctionetc.), renal tubular affections, circulatory affections,encephalopathies (apoplexy etc.) and nervous diseases), immunity-relateddiseases such as acquired immunodeficiency syndrome, affections ofthymocyte caused by administration of immunosuppressive agent oranticancer agent, decrease of peripheral T cells and immune deficiency,inflammations such as hepatitis and ulcerative colitis, externalinjuries, bacterial infections, viral infections, Alzheimer's disease,diabetes mellitus, auxeses, Kawasaki disease, schizophrenia,fervescence, metabolic diseases, cancers and so forth.

Moreover, the Hsp expression promoter can be used not only for diseasescaused by cell injury or cell death, but also for objectives for whichcell or tissue protective action can be expected. In this respect,detailed explanation will be made in the section of the “cell and tissueprotecting agent” described later.

A subjective animal to be administered with the Hsp expression promoteris preferably a vertebrate, especially a mammal, particularly preferablya human. Although the Hsp expression promoter can be administered forthe purpose of prevention of those diseases or suppression of advance ofthose diseases (prevention of aggravation), amelioration or treatment ofsymptoms and so forth, it is preferably administered as a drug forprophylactic treatment.

The formulation amounts, doses for single administration, administrationintervals and so forth of the HA oligosaccharides, especially HAtetrasaccharide, contained in the Hsp expression promoter are factors tobe individually determined depending on the administration route, dosageform, purpose of use etc. of the promoter as well as specific symptoms,age, sex, body weight of patients, and they are not particularlylimited. However, the clinical dose of HA tetrasaccharide may be, forexample, 300 to 7500 mg per single administration for an adult. As forthe administration interval of the Hsp expression promoter, it may beadministered one time per day, or dividedly administered 2 to 3 timesper day.

The Hsp expression promoter can be used also as a test reagent for anexperiment concerning the stress protein expression promoting action.

[2] Cell Death Inhibitor

The cell death inhibitor constitutes one of the uses utilizing the Hspexpression promoting action. That is, since HA oligosaccharides(especially HA tetrasaccharide) not only exhibit the Hsp expressionpromoting action but also actually exhibit cell death inhibitory effect,it is used for the purpose of suppression of cell death.

Diseases for which the cell death inhibitor can be used are notparticularly limited so long as they are diseases in which cell deathcan be inhibited by enhancement of Hsp expression. However, the diseasesexemplified in the explanation of the Hsp expression promoter arepreferably mentioned.

The animals to be administered with this cell death inhibitor, purposeof administration, formulation amounts, doses, administration intervalsof the HA oligosaccharides (especially HA tetrasaccharide) are similarto those mentioned for the Hsp expression promoter.

[3] Cell Injury Inhibitor

Since the HA oligosaccharides (especially HA tetrasaccharide) alsoactually exhibit cell injury inhibitory action in addition to the Hspexpression promoting action, they are applied for the purpose ofsuppression of cell injury.

Diseases for which the cell injury inhibitor can be used are notparticularly limited so long as they are diseases in which in which cellinjury can be inhibited by enhancement of Hsp expression. However, thediseases exemplified in the explanation of the Hsp expression promoterare preferably mentioned.

The animals to be administered with this cell injury inhibitor, purposeof administration, formulation amounts, doses, administration intervalsetc. of the HA oligosaccharides (especially HA tetrasaccharide) aresimilar to those mentioned for the Hsp expression promoter.

[4] Cell and Tissue Protecting Agent

Since the HA oligosaccharides (especially HA tetrasaccharide) alsoactually exhibit cell and tissue protecting action in addition to theHsp expression promoting action, they are applied for the purpose ofcell and tissue protection.

This cell and tissue protecting agent can be used for objectives forwhich cell and tissue protecting action by Hsp is expected. For example,it can be used for diseases for which cell or tissue protection isdesired, protection of cells and tissues extracted out of organisms andso forth.

Examples of the diseases for which protection of cells or tissues isdesired include peptic ulcers (gastric ulcer, duodenal ulcer etc.),gastritis, hepatopathies (hepatitis etc.), ulcerative colitis, ischemicheart diseases, myocardosis, apoplexy, cerebral infarction and so forth.

Specifically, this cell and tissue protecting agent is preferably usedas the following agents.

(1) Antiulcer Agent and Antihepatopathic Agent

By using the aforementioned cell and tissue protecting agent as an agentfor treatment of ulcer, it can be an antiulcer agent. By using theaforementioned cell and tissue protecting agent as an agent fortreatment of hepatopathy, it can be an antihepatopathic agent. The“treatment” used in the present invention means administration of anagent to an animal with a disease of interest with a purpose ofprevention, maintenance (prevention of aggravation), amelioration(improvement of symptoms) or cure of the disease, and the term “agentfor treatment” means an agent used for such a treatment.

(2) IL-10 Production Promoter and IL-8 Production Inhibitor

The aforementioned cell and tissue protecting agent can also be used fortreatment of diseases caused by decrease of IL-10, diseases for whichpromotion of IL-10 production is desired and so forth. By using the celland tissue protecting agent as an agent for promoting IL-10 production,it can be an IL-10 production promoter.

The aforementioned cell and tissue protecting agent can also be used fortreatment of diseases caused by increase of IL-8, diseases for whichinhibition of IL-8 production is desired and so forth. By using the celland tissue protecting agent as an agent for inhibiting IL-8 production,it can be an IL-8 production inhibitor.

Animals that can be objectives of these agents, purposes ofadministration thereof and so forth are similar to those mentioned inthe explanation of the Hsp expression promoter.

(3) Organ Preservation Agent

The aforementioned cell and tissue protecting agent can also be used forthe purpose of protecting cells and tissues extracted out of organismsand so forth. By using the cell and tissue protecting agent as an agentfor preservation of cells and tissues extracted out of organisms, it canbe, for example, an organ preservation agent. Specifically, by perfusingan organ removed for transplantation (liver, kidney, heart, lung etc.)with the organ preservation agent, or storing or maintaining such anorgan in the organ preservation agent, cell or tissue affections of suchan organ (edema, cell death etc.) can be inhibited.

The formulation amounts, doses for single administration (use),administration intervals of the HA oligosaccharides contained in theorgan preservation agent can be individually determined depending onspecific situations of administration such as administration route(method of use), dosage form, purpose of use and so forth.

For example, when the cell and tissue protecting agent is administeredto a human as an antiulcer agent, antihepatopathic agent, IL-10production promoter or IL-8 production inhibitor, the clinical dose maybe 120 to 6000 mg for single dose for an adult as amount of HAoligosaccharides (especially HA tetrasaccharide).

When the cell and tissue protecting agent is used for the purpose ofprotecting cells or tissues extracted out of an organism, it may beformulated as a liquid preparation, perfusion, solid preparation fordissolution upon use or the like. When it is used as the organpreservation agent, the formulation amounts of the HA oligosaccharides(especially HA tetrasaccharide) and so forth may be individuallydetermined depending on specific conditions such as size of organ to bestored or maintained, preservation time and temperature.

Although the concentration of the HA oligosaccharide (especially HAtetrasaccharide) in the cell and tissue protecting agent is not alsoparticularly limited, when it is provided as an injection (solution) ora liquid preparation for organ preservation, it may be preferably 1ng/mL to 1 mg/mL.

When the cell and tissue protecting agent is provided as an injection ora liquid preparation, it may be in the form of solution, frozen product,lyophilized product or the like. These may be filled and sealed in anappropriate container such as ampoule, vial, syringe for injection andbottle, distributed or stored as they are, and used as an injection orliquid preparation.

The present invention also provides, in addition to the agents mentionedabove, a method for promoting Hsp expression, inhibiting cell death,inhibiting cell injury, or protecting cell and tissue (e.g., preservingan organ, treating an ulcer, treating a hepatopathy, promoting IL-10production or inhibiting IL-8 production) in an objective of applicationsuch as cells and biological tissues, which comprises allowing the HAoligosaccharide (especially HA tetrasaccharide) to act on the object ofthe application in vitro or in vivo.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows an elution curve of HA4 in gel filtration chromatography.The ordinate of the upper graph represents absorbance at 210 nm, and theordinate of the lower graph represents absorbance of RI. Further, eachabscissa represents elution time.

FIG. 2 shows an elution curve of HA6 in gel filtration chromatography.The ordinates and abscissas have the same meanings as in FIG. 1.

FIG. 3 shows an elution curve of HA8 in gel filtration chromatography.The ordinates and abscissas have the same meanings as in FIG. 1.

FIG. 4 shows an elution curve of HA10 in gel filtration chromatography.The ordinates and abscissas have the same meanings as in FIG. 1.

FIG. 5 shows an elution curve of HA12 in gel filtration chromatography.The ordinates and abscissas have the same meanings as in FIG. 1.

FIG. 6 shows an elution curve of HA14 in gel filtration chromatography.The ordinates and abscissas have the same meanings as in FIG. 1.

FIG. 7 shows an elution curve of HA4 in anion exchange chromatography.The ordinate represents absorbance at 210 nm, and the abscissarepresents elution time.

FIG. 8 shows an elution curve of HA6 in anion exchange chromatography.The ordinate and abscissa have the same meanings as in FIG. 7.

FIG. 9 shows an elution curve of HA8 in anion exchange chromatography.The ordinate and abscissa have the same meanings as in FIG. 7.

FIG. 10 shows an elution curve of HA10 in anion exchange chromatography.The ordinate and abscissa have the same meanings as in FIG. 7.

FIG. 11 shows an elution curve of HA12 in anion exchange chromatography.The ordinate and abscissa have the same meanings as in FIG. 7.

FIG. 12 shows an elution curve of HA14 in anion exchange chromatography.The ordinate and abscissa have the same meanings as in FIG. 7.

FIG. 13 shows an elution curve of HA48 in anion exchange chromatography.The ordinate and abscissa have the same meanings as in FIG. 7.

FIG. 14 shows an elution curve of HA50 in anion exchange chromatography.The ordinate and abscissa have the same meanings as in FIG. 7.

FIG. 15 shows an elution curve of HA52 in anion exchange chromatography.The ordinate and abscissa have the same meanings as in FIG. 7.

FIG. 16 shows results of electrophoresis of fluorescence-labeledoligosaccharides.

FIG. 17 shows mass spectrum of HA4 (Lot 1).

FIG. 18 shows mass spectra of HA6 (upper graph), HA8 (middle graph) andHA10 (lower graph).

FIG. 19 shows results of deconvolution of HA6 (upper graph), HA8 (middlegraph) and HA10 (lower graph).

FIG. 20 shows mass spectra of HA12 (upper graph), HA14 (middle graph)and HA16 (lower graph).

FIG. 21 shows the result of deconvolution of HA12 (upper graph), HA14(middle graph) and HA16 (lower graph).

FIG. 22 shows mass spectra of HA48 (upper graph), HA50 (middle graph)and HA52 (lower graph).

FIG. 23 shows results of deconvolution of HA48 (upper graph), HA50(middle graph) and HA52 (lower graph).

FIG. 24 shows ¹H-NMR spectrum of HA4.

FIG. 25 shows ¹³C-NMR spectrum of HA4.

FIG. 26 shows ¹H-NMR spectrum of HA6.

FIG. 27 shows ¹³C-NMR spectrum of HA6.

FIG. 28 shows ¹H-NMR spectrum of HA8.

FIG. 29 shows ¹³C-NMR spectrum of HA8.

FIG. 30 shows ¹H-NMR spectrum of HA10.

FIG. 31 shows ¹³C-NMR spectrum of HA10.

FIG. 32 shows ¹H-NMR spectrum of HA12.

FIG. 33 shows ¹³C-NMR spectrum of HA12.

FIG. 34 shows ¹H-NMR spectrum of HA14.

FIG. 35 shows ¹³C-NMR spectrum of HA14.

FIG. 36 shows ¹H-NMR spectrum of HA16.

FIG. 37 shows ¹³C-NMR spectrum of HA16.

FIG. 38 shows ¹H-NMR spectrum of HA48.

FIG. 39 shows ¹H-NMR spectrum of HA50.

FIG. 40 shows ¹H-NMR spectrum of HA52.

FIG. 41 shows activation of HSF1 by HA tetrasaccharide after heat shock.

FIG. 42 shows expression of Hsp72 induced by HA tetrasaccharide afterheat shock.

FIG. 43 shows degrees of suppression of cell death (apoptosis) inducedby serum starvation obtained with various HA oligosaccharides.

FIG. 44 shows edema suppression effect of HA tetrasaccharide in anorgan.

FIG. 45 shows cellular chromatin concentration inhibitory effect of HAtetrasaccharide.

FIG. 46 shows decrease of TUNEL stained cell number obtained with HAtetrasaccharide.

FIG. 47 shows cell and tissue affection inhibitory effect of HAtetrasaccharide.

FIG. 48 shows gastric ulcer inhibitory effect of HA tetrasaccharide.

FIG. 49 shows hepatopathy inhibitory effect (GOT activity) of HAtetrasaccharide.

FIG. 50 shows hepatopathy inhibitory effect (leucocyte count) of HAtetrasaccharide.

FIG. 51 shows IL-10 production promotion effect of HA tetrasaccharide.

FIG. 52 shows IL-8 production inhibitory effect of HA tetrasaccharide.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained more specificallywith reference to the following examples.

Hereafter, hyaluronic acid oligosaccharide fractions are referred towith abbreviations of “HA4” for HA tetrasaccharide fraction, “HA6” forHA hexasaccharide fraction, “HA8” for HA octasaccharide fraction etc.

Example 1 Preparation and Physicochemical Properties of Oligosaccharidesand Fractions of the Present Invention 1. Preparation ofOligosaccharides and Fractions of the Present Invention

Oligosaccharides and fractions of the present invention were prepared bythe following procedures using sodium salt of HA isolated and purifiedfrom chicken crest as a raw material. The sodium salt of HA used as theraw material showed a single band in electrophoresis using a celluloseacetate membrane (electrophoresis buffer: pyridine/formic acid buffer,electric current: 15 mA, and migration time: 30 minutes), andglycosaminoglycans other than HA (chondroitin, chondroitin-4-sulfate,chondroitin-6-sulfate, chondroitin sulfate E, chondroitin sulfate D,heparin, heparan sulfate, dermatan sulfate) were not detected.

Preparation Example 1 Decomposition by Hyaluronidase

In an amount of 25 g of the sodium salt of HA was dissolved in 1.1 L of0.1 M phosphate buffer (pH 5.3) containing 0.15 M NaCl. To thissolution, 200 mg of hyaluronidase derived from bovine testis (5.342units/mg, produced by Seikagaku Corporation) was added and allowed toreact at 37° C. for 9 hours.

After the reaction, the reaction mixture was centrifuged at 10,000 rpmfor 30 minutes, and the supernatant was collected. The collectedsupernatant was applied to an ion exchange column (φ1.5×123 cm) of astrongly basic anion exchanger having trimethylammoniomethyl groups asanion exchange groups, Dowex 1×2 (100-200 mesh, produced by DowChemical) and eluted with a linear concentration gradient of NaCl (0.01M to 0.50 M). Uronic acid in the obtained fractions was detected by thecarbazole method to screen fractions containing HA oligosaccharides.Appropriate fractions were pooled and concentrated, and after desaltingwith Sephadex G-10 (produced by Pharmacia, φ3×124), the concentrate waslyophilized.

Weights of the lyophilized products of the obtained fractions are shownin the parentheses.

HA4 (330 mg), HA6 (1210 mg), HA8 (305 mg), HA10 (1625 mg), HA12 (685mg), HA14 (620 mg), HA16 (430 mg), HA18 (210 mg), HA20 (202 mg), HA22(819 mg), HA24 (197 mg), HA26 (187 mg), HA28 (159 mg), HA30 (137 mg),HA32 (122 mg), HA34 (102 mg), HA36 (91 mg), HA38 (89 mg), HA40 (65 mg),HA42 (76 mg), HA44 (61 mg), HA46 (58 mg), HA48 (46 mg), HA50 (48 mg),HA52 (21 mg)

Preparation Example 2 Decomposition by Chondroitinase ACI

In an amount of 8 g of the sodium salt of HA was dissolved in 500 mL of0.1 M acetate buffer (pH 6.0) containing 0.1% bovine serum albumin(BSA).

To this solution, 32 units of chondroitinase ACI (produced by SeikagakuCorporation) was added and allowed to react at 37° C. for 6 hours.

After the reaction, the reaction mixture was centrifuged at 10,000 rpmfor 30 minutes, and the supernatant was collected. The collectedsupernatant was applied to an ion exchange column (φ4.5×123 cm) of Dowex1×2 (100-200 mesh, produced by Dow Chemical) and eluted with a linearconcentration gradient of NaCl (0.01 M to 0.50 M). Uronic acid in theobtained fractions was detected by the carbazole method to screenfractions containing HA oligosaccharides. Appropriate fractions werepooled and concentrated, and after desalting with Sephadex G-10(produced by Pharmacia, φ3×124), the concentrate was lyophilized.

Weights of the lyophilized products of the obtained fractions are shownin the parentheses. “Δ” means that the saccharide at the non-reducingend is an unsaturated saccharide.

ΔHA4 (133 mg), ΔHA6 (133 mg), ΔHA8 (84 mg), ΔHA10 (109 mg), ΔHA12 (100mg), ΔHA14 (101 mg), ΔHA16 (73 mg), ΔHA18 (31 mg), ΔHA20 (9 mg)

Preparation Example 3 Dimethyl Sulfoxide (DMSO) Method

In an amount of 10 g of the sodium salt of HA was dissolved in 3 L ofdimethyl sulfoxide containing 10% of 0.1 M HCl and subjected to a heattreatment at 105° C. for 16 hours.

After the treatment, the obtained solution was applied to Dowex 1×2(100-200 mesh, produced by Dow Chemical) ion exchange column (φ3.0×78cm) to perform chromatography. Elution was performed with a linearconcentration gradient of NaCl (0.01 M to 0.50 M). Uronic acid in theobtained fractions was detected by the carbazole method to screenfractions containing HA oligosaccharides. Appropriate fractions werepooled and concentrated, and after desalting with Sephadex G-10(produced by Pharmacia, φ3×124), the concentrate was lyophilized.

Weights of the lyophilized products of the obtained fractions are shownin the parentheses.

HA2 (50 mg), HA4 (1100 mg), HA6 (232 mg), HA8 (1015 mg), HA10 (1033 mg),HA12 (459 mg)

The HA oligosaccharides of various sizes obtained as described abovewere used for the following various analyses.

2. Physicochemical Properties of Fractions of the Present Invention

Various physicochemical properties of each of the HA oligosaccharide(sodium salt) fractions obtained in Preparation Example 1 mentionedabove were investigated.

(1) Analysis by gel filtration chromatographyUsed column: TSK Gel 2500+3000+4000 pwxl (TOSOH)

Solvent: 0.05 M NaCl

Flow rate: 0.6 mL/minuteDetection wavelength: 210 nm, differential refractometer (RI)Applied sample amount: 200 μg/shot as amount of HA oligosaccharide

The elution curves obtained by using HA4 (Lot 1) to HA14 as samples areshown in FIGS. 1 to 6. In each figure, the upper graph indicates theresult of detection based on the absorbance at 210 nm, and the lowergraph indicates the result of detection by using a differentialrefractometer.

From the results shown in these figures, it can be seen that all thefractions showed a substantially single peak.

Further, for all the cases, the relative area of the main HA peak to thesum of the peak areas of the total HA oligosaccharides in each fractionwas 85% or more as detected based on the absorbance at 210 nm or 98% ormore as detected by using a differential refractometer (RI) as shown inTable 1.

TABLE 1 Ratio of main peak area to total peak areas (%) Sample 210 nm RIHA4 (Lot 1) 89.037 99.854 HA6 98.573 99.815 HA8 96.617 98.929 HA1098.594 99.287 HA12 100.000 100.000 HA14 98.334 99.796

(2) Analysis by Ion Exchange Chromatography

Column: YMC NH2 column (YMC)Solvent: Concentration gradient of NaH₂PO₄ from 0 M to 0.8 MFlow rate: 1 mL/minuteDetection wavelength: 210 nmApplied sample amount: 20 μg/shot as amount of HA oligosaccharide

The elution curves obtained by using HA4 (Lot 1) to HA14 and HA48 toHA52 as samples are shown in FIGS. 7 to 15. From the results shown inthese figures, it can be seen that all the fractions showed asubstantially single peak. Further, for all the cases, the relative areaof the main HA peak to the sum of the peak areas of the total HAoligosaccharides in each fraction was 90% or more as shown in Table 2.

TABLE 2 Ratio of main peak area to total peak Sample areas (%) HA4(Lot 1) 98.5007 HA6 97.9140 HA8 98.1209 HA10 97.4236 HA12 94.5425 HA1494.7181 HA48 94.4743 HA50 93.4305 HA52 94.2492

(3) Analysis by Fluorescence-Labeled Gel Electrophoresis

Each of HA4, HA6, HA8, HA10, HA12, HA14 and HA16 was used to perform thefollowing procedure. At the same time, a fraction of HA oligosaccharidemixture (also containing HA disaccharide, referred to as the “mixture”hereinafter) was also used to perform the same procedure.

(3-1) Preparation of Fluorescence-Labeled HA Oligosaccharides

HA oligosaccharides contained in each fraction were labeled withfluorescence by using FACE® N-linked oligosaccharide Profiling Kit(Glyko, Inc., Novato, Calif., U.S.A.).

A fraction in such an amount that the fraction should contain about 2nmol of HA oligosaccharides (in the case of the mixture, about 20 nmolof oligosaccharides as HA disaccharide units) was lyophilized in a0.5-mL plastic tube by using a centrifugation type vacuum lyophilizer(SpeedVac, AS160, SAVANT INSTRUMENTS INC., NY, U.S.A). After thelyophilization, 5 μL of 8-aminonaphthalene-1,3,6-trisulfonic aciddisodium salt (ANTS) solution (GLYKO, L2, Part #50058: reconstitutedOLIGO Labeling Dye) was added to each tube, stirred and left at roomtemperature for 15 minutes. Furthermore, 5 μL of sodium cyanoborohydridesolution (GLYKO, L1, Part #50056: Labeling Reducing Agent) was added toeach tube, and the tube was sealed and incubated at 36° C. for 16 hours.

After the incubation, the mixture was partially dried in acentrifugation type vacuum lyophilizer for about 15 minutes and thenadded with water to a volume of 20 μL.

(3-2) Electrophoresis of Fluorescence-Labeled Oligosaccharides

Electrophoresis was performed by using GLYKO O-linked OligosaccharideProfiling Gel (GLYKO, Part #60200: polyacrylamide gel having acrosslinking degree of 36%).

One pack of OLIGO Gel Running Buffer (GLYKO, Part #7000) was dissolvedin distilled water so that the solution should have a volume of 1.5 Land cooled on ice.

Cooling water was circulated in a gel box (GLYKO, GLYKO Gel Box, Part#40026), the cooled Running Buffer was placed in it, and the gel was seton it. The gel box was placed on ice for cooling.

Each fraction solution containing fluorescence-labeled HAoligosaccharides was taken in a volume of 2 μL and mixed with 3 μL ofdistilled water and 5 μL of Loading Buffer (GLYKO, Part #50064). As forthe mixture solution, 2 μL of the solution was mixed with 2 μL ofLoading Buffer. The mixture in a volume of 4 μL was applied to the gel.Each fraction was applied in such an amount that 80 pmol of HAoligosaccharides should be applied per lane.

Electrophoresis was performed with a constant voltage of 1000 V for 160minutes, and fluorescence emitted upon irradiation of a light at 365 nmlight from an UV transilluminator (Model NLM-20E, UVP, CA, U.S.A.) wasrecorded by using an instant camera (MAMIYA, Professional SD, f=127 mm)and an instant film (Polaloid® Polapan T667, ISO 3000, Polapoid Corp.,MA, U.S.A). The results are shown in FIG. 16.

The relationships of the lanes in FIG. 16 and the samples are asfollows.

Lane 1: mixture, Lane 2: HA4, Lane 3: HA6, Lane 4: HA8, Lane 5: HA10,Lane 6: HA12, Lane 7: HA14, Lane 8: HA16

While the mixture showed ladder-like bands, all of the fractionatedoligosaccharide fractions each formed a single band with migrationcorresponding to the size of each oligosaccharide, and bands of HAoligosaccharides of other sizes were not detected.

Further, an oligosaccharide of a smaller size showed a larger migrationdegree. The positions of bands (migration position) observed for thefractions well corresponded to those of the ladder-like bands observedfor the mixture, and thus it was demonstrated that the migration degreesof molecules were not affected even in a mixture of oligosaccharides ofmultiple kinds of sizes.

GLYKO O-linked Profiling Gel is polyacrylamide gel having a crosslinkingdegree of 36%. 8-Aminonaphthalene-1,3,6-trisulfonic acid disodium salt(ANTS) is a fluorescent substance having trivalent negative charge.

When 2-aminoacridon (AMAC) having no charge was used as a fluorescentsubstance, all of the oligosaccharides did not substantially migrate onthis gel. This suggested that only the negative charge of HAoligosaccharide itself was insufficient for migration on the gel of highcrosslinking degree, and charge of bound ANTS was required. Further,since boric acid known to interact with a saccharide chain basicstructure was not used, it was suggested that the relationship betweenthe oligosaccharide size and the mesh size of polyacrylamide directlydetermine the migration degree.

When other gels available from Glyko (crosslinking degree of 20% or 21%)and labeling with ANTS were used, HA octasaccharide and oligosaccharidesof smaller sizes could not be separated, or HA tetrasaccharide andoligosaccharides of smaller sizes could not be detected because theyoverlapped with the migration front.

(4) Mass Spectrometry

Mass spectrometry was performed by the electrospray ionization massspectrometry (ESIMS) method.

(4-1) Method (4-1-1) Sample for Analysis

Aqueous solutions of HA4 (Lot 1), HA6, HA8, HA10, HA12, HA14, HA16,HA48, HA50 and HA52 were prepared to have concentrations of 2.6 mg/mL,1.2 mg/mL, 1.4 mg/mL, 3.0 mg/mL, 2.0 mg/mL, 6.5 mg/mL, 1.3 mg/mL, 1mg/mL, 1 mg/mL and 1 mg/mL, respectively, and analyzed.

The theoretical molecular weights of HA oligosaccharides were obtainedbased on the fact that HA tetrasaccharide to HA dopentacontasaccharidehave a structure represented by the following formula (1).

(In the formula, n is an integer of 1 to 29, M represents a proton or amonovalent cation, and Ac represents acetyl group.)

(4-1-2) Reagents

Methanol (Wako Pure Chemical Industries) and distilled water (Wako PureChemical Industries) were those for HPLC, and ammonium formate ofspecial grade (Wako Pure Chemical Industries) was used.

(4-1-3) Instruments and Equipments 1) HPLC System

Agilent 1100 Series: binary pump, degasser, autosampler (AgilentTechnologies)

2) Mass Spectrometer

Triple quadrupole mass spectrometer: TSQ (Thermoquest)

(4-1-4) ESIMS Analysis Conditions

Introduction of a sample into the mass spectrometer was attained byinjecting 5 μL or 10 μL of each sample solution into the HPLC systemconnected to the mass spectrometer. The analysis conditions of HPLC andESIMS are as follows.

1) HPLC

Mobile phase: 10 mM ammonium formate aqueoussolution/methanol=80/20Flow rate: 0.2 mL/minuteColumn: not usedInjection amount: 10 μL

2) ESIMS Probe: Off-axis

Ion mode: anion modeIonizing method: ESI method (electrospray ionization method)ESI spray voltage: 4.5 kVHeated capillary temperature: 350° C.Auxiliary gas: 35 units

Sheath gas: 50 psi

Scanning range: m/z 10-2500 or 10-4000Scanning time: 1.5 seconds or 3 seconds

(4-1-5) Deconvolution Analysis

Deconvolution analysis for estimation of molecular weight from anobserved ESIMS spectrum was performed by using analysis software,Xcalibur Bioworks (ThermoQuest).

(4-2) Results

The results of the anion ESIMS spectrum measurement of HA4 to HA16 aresummarized in Tables 3 and 4, and spectra of HA4 to HA16 and HA48 toHA52 are shown in FIGS. 17 to 23.

TABLE 3 Pseudomolecule Ions And Related Ions Thereof (m/z) Sample [M—H]⁻[M—2H]²⁻ [M—3H]³⁻ [M—4H]⁴⁻ [M—5H]⁵⁻ [M + Na—2H]⁻ [M + Na—3H]²⁻ [M +2Na—4H]²⁻ HA4 774.9 386.9 797.1 (Lot1) 100 55 27 HA6 1153.9 576.6 383.81176.1 587.6 45 100 1 2 19 HA8 1533.9 766.1 510.3 777.0 36 100 3 9 HA101912.8 955.9 636.9 967.0 16 100 15 4 HA12 2292.1 1145.5 763.2 572.31156.3 1167.4 12 100 91 13 16 8 HA14 1335.3 889.5 666.6 1346.2 1357.5 79100 12 9 4 HA16 1524.8 1016.1 761.6 608.9 1535.7 52 100 17 5 4Pseudomolecule Ions And Related Ions Thereof (m/z) Deconvolution Sample[M + Na—4H]³⁻ [M + 2Na—5H]³⁻ [M + 3Na—OH]³⁻ [2M—H]⁻ [2M—3H]³⁻ mass HA41551.6 15 HA6 769.1 1155.1 3 HA8 1022.3 1534.5 10 HA10 1275.2 1913.7 9HA12 770.5 777.9 1527.9 2293.1 15 8 11 HA14 897.1 1780.6 2671.9 14 4HA16 1023.4 1030.6 1037.6 3051.2 16 9 3 Upper Line: Measured Value LowerLine: Relative Intensity of Peak(%)

TABLE 4 Molecule Related Ions(m/z) Sample [M—5H]⁵⁻ [M—6H]⁶⁻ [M—7H]⁷⁻[M—8H]⁸⁻ [M—9H]⁹⁻ [M—10H]¹⁰⁻ [M—11H]¹¹⁻ [M—12H]¹²⁻ HA48 1822.4 1518.71301.9 1138.4 1011.7 910.5 827.9 758.6 100 7 6 12 16 37 77 59 HA501898.2 1581.4 1355.5 1186.5 1054.1 948.7 861.9 790.3 100 7 2 7 8 16 2623 HA52 1974.2 1645.3 1409.5 1233.0 1096.4 986.7 896.6 821.8 100 23 7 1123 27 69 98 Molecule Related Ions(m/z) Deconvolution Sample [M—13H]¹³⁻[M—14H]¹⁴⁻ [M + Na—6H]⁶⁻ [M + 2Na—7H]⁵⁻ [M + 3Na—8H]⁵⁻ mass HA48 700.2650.0 1826.7 1831.7 1835.6 9117.2 22 8 45 25 23 HA50 729.4 677.1 1903.01907.3 1911.8 9496.1 19 14 43 19 12 HA52 758.4 704.3 1979.3 1983.51987.4 9875.0 45 20 50 37 39 Upper Line: Measured Value Lower Line:Relative Intensity of Peak(%)

In the aforementioned results, various molecule-related ions allconsidered to be derived from HA oligosaccharides were observed, andthey are not contradictory to interpretations that HA4 (Lot 1) shouldcorrespond to HA tetrasaccharide, HA6 to hexasaccharide, HA8 to HAoctasaccharide, HA10 to HA decasaccharide, HA12 to HA dodecasaccharide,HA14 to HA tetradecasaccharide, HA16 to HA hexadecasaccharide, HA48 toHA octatetracontasaccharide, HA50 to HA pentacontasaccharide and HA52 toHA dopentacontasaccharide.

Based on the above results, relative values of the actually measuredvalues for the main peaks with respect to the theoretical values ofmonoisotopic molecular weights, which were taken as 1, were calculated.The results are shown in Table 5A. In addition, as for HA4, the actuallymeasured value and theoretical value for [M-H]⁻ were used.

TABLE 5A Sample Found Calculated Relative value HA4 (Lot 1) 774.9 775.20.9996 HA6 1155.1 1155.3 0.9998 HA8 1534.5 1534.5 1.0000 HA10 1913.71913.6 1.0001 HA12 2293.1 2292.7 1.0002 HA14 2671.9 2671.8 1.0000 HA163051.2 3050.9 1.0001 HA48 9117.2 9116.7 1.0001 HA50 9496.1 9495.8 1.0000HA52 9875.0 9874.9 1.0000

Further, relative values of the actually measured values for the mainpeaks with respect to the theoretical values of average molecularweights, which are taken as 1, were calculated. The results are shown inTable 5B. As for HA4, the actually measured value and theoretical valuefor [M-H]⁻ were used.

TABLE 5B Sample Found Calculated Relative value HA4 (Lot 1) 774.9 775.60.9991 HA6 1155.1 1156.0 0.9992 HA8 1534.5 1535.3 0.9995 HA10 1913.71914.6 0.9995 HA12 2293.1 2293.9 0.9997 HA14 2671.9 2673.3 0.9995 HA163051.2 3052.6 0.9995 HA48 9117.2 9121.7 0.9995 HA50 9496.1 9501.0 0.9995HA52 9875.0 9880.3 0.9995

From these results, it was demonstrated that, when the theoreticalvalues of the monoisotopic molecular weights or average molecularweights of HA oligosaccharides constituting the fractions of the presentinvention were taken as 1, the actually measured values of the fractionsobtained by mass spectrometry were within the range of 0.999-1.001(relative value).

(5) Elemental Analysis

HA4 (Lot 1), HA6, HA8, HA10, HA12, HA14 and HA16 were dried at 80° C.for 2 hours under reduced pressure and immediately analyzed by using anelemental analyser. The results are shown in Table 6.

TABLE 6 Sample Element Calculated (%) Found (%) Error HA4 (Lot 1) C40.98 40.63 0.35 H 5.16 5.20 −0.04 N 3.41 3.34 0.07 Na 5.60 5.48 0.12HA6 C 41.28 40.74 0.54 H 5.11 5.17 −0.06 N 3.44 3.31 0.13 Na 5.64 5.75−0.11 HA8 C 41.44 41.41 0.03 H 5.09 5.22 −0.13 N 3.45 3.36 0.09 Na 5.675.41 0.26 HA10 C 41.53 41.41 0.12 H 5.08 5.24 −0.16 N 3.46 3.43 0.03 Na5.68 5.45 0.23 HA12 C 41.59 40.96 0.63 H 5.07 5.33 −0.26 N 3.46 3.230.23 Na 5.69 5.41 0.28 HA14 C 41.64 41.44 0.20 H 5.06 5.36 −0.30 N 3.473.37 0.10 Na 5.69 5.20 0.49

Thus, it was demonstrated that the differences between the theoreticalvalues of content ratios (weight %) of carbon (C), hydrogen (H),nitrogen (N) and sodium (Na) in HA oligosaccharides (sodium salts)constituting the fractions of the present invention and the actuallymeasured values of the same (weight %) obtained by elemental analysis ofthe corresponding fractions were all with in the range of ±1 (weight %).

(6) Nuclear Magnetic Resonance Spectrum (NMR)

¹H-NMR and ¹³C-NMR of the HA oligosaccharide fractions were measured byusing VARIAN UnityInova Model 500. As the measurement solvent, D₂O wasused. The measurement was performed by using t-BuOH (¹H, 1.23 ppm, ¹³C,32.461 ppm) as an internal standard at a measurement temperature of 23°C. The results for the HA oligosaccharide fractions are shown below.Further, spectra of the fractions are shown in FIGS. 24 to 40.

(6-1) Measurement Result of HA4 (Lot 1)

500 MHz ¹H-NMR δ; 2.004 (s, 3H, NAc), 2.018, 2.019 (3H, NAc),3.292-3.326 (m, 1H, H-2d), 3.354 (dd, 1H, J_(1,2)=7.9 Hz, J_(2,3)=9.5Hz, H-2b), 4.027 (dd, 0.6H, J_(1,2)=3.5 Hz, J_(2,3)=10.6 Hz, H-2aα),4.453 (d, J_(1,2)=7.9 Hz, H-1d), 4.460 (d, H-1bβ), 4.502 (d, 0.6H,H-1bα), 4.555 (d, 1H, J_(1,2)=8.4 Hz, H-1c), 4.705 (d, 0.4H, J_(1,2)=8.4Hz, H-1aβ), 5.144 (d, 0.6H, H-1aα).

The spectrum used as the basis of the analysis is shown in FIG. 24.

125 MHz ¹³C-NMR δ; 24.84, 25.09, 25.36 (NHCOCH₃), 55.81 (C-2aα), 57.09(C-2c), 58.44 (C-2aβ), 63.40, 63.57 (C-6a or C-6c), 75.29, 75.33 (C-2bαor C-2bβ), 75.58 (C-2d), 93.92 (C-1aα), 97.61 (C-1aβ), 103.44 (C-1c),105.80, 105.84, 105.95 (C-1bα or C-1bβ or C-1d), 177.00, 177.04, 177.41,177.68, 177.80, 178.33 (carbonyl).

The spectrum used as the basis of the analysis is shown in FIG. 25.

These results are not contradictory to the structure represented by theaforementioned formula (1) wherein n=1 (structure of sodiumβ-D-glucopyranosyluronate-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-(sodiumβ-D-glucopyranosyluronate)-(1→3)-2-acetamido-2-deoxy-D-glucopyranose).

(6-2) Measurement Results of HA6

500 MHz ¹H-NMR δ; 2.002, 2.010, 2.017 (9H, NAc), 3.291-3.368 (m, 3H,H-2b, H-2d and H-2f), 4.026 (dd, 0.6H, J_(1,2)=3.5 Hz, J_(2,3)=10.6 Hz,H-2aα), 4.452, 4.459 (d×2, 2.4H, J_(1,2)=7.8 Hz, J_(1,2)=7.8 Hz, H-1bβ,H-1d and H-1f), 4.500 (d, 0.6H, J_(1,2)=7.9 Hz, H-1bα), 4.544, 4.550(d×2, 2H, J_(1,2)=8.5 Hz, J_(1,2)=8.5 Hz, H-1c or H-1e), 4.702 (d,J_(1,2)=8.3 Hz, H-1aβ), 5.142 (d, 0.6H, H-1aα).

The spectrum used as the basis of the analysis is shown in FIG. 26.

125 MHz ¹³C-NMR δ; 24.84, 25.09, 25.35 (NHCOCH₃), 55.81 (C-2aα), 57.09,57.16 (C-2c or C-2e), 58.44 (C-2aβ), 63.40, 63.57 (C-6a or C-6c orC-6e), 75.28, 75.31, 75.34, 75.57 (C-2bα or C-2bβ or C-2d or C-2f),93.92 (C-1aα), 97.61 (C-1aβ), 103.40, 103.45 (C-1c or C-1e), 105.80,105.85, 105.94, 106.02 (C-1bα or C-1bβ or C-1d or C-1f), 176.97, 177.40,177.68, 177.80, 178.27 (carbonyl).

The spectrum used as the basis of the analysis is shown in FIG. 27.

These results are not contradictory to the structure represented by theaforementioned formula (1) wherein n=2 (structure of sodiumβ-D-glucopyranosyluronate-[(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-(sodiumβ-D-glucopyranosyluronate)]₂-(1→3)-2-acetamido-2-deoxy-D-glucopyranose).

(6-3) Measurement Results of HA8

500 MHz ¹H-NMR δ; 2.002, 2.003, 2.008, 2.016 (12H, NAc), 3.290-3.368 (m,4H, H-2b, H-2d, H-2f and H-2h), 4.025 (dd, 0.6H, J_(1,2)=3.5 Hz,J_(2,3)=10.6 Hz, H-2aα), 4.450, 4.458 (d×2, 3.4H, J_(1,2)=7.8 Hz,J_(1,2)=7.8 Hz, H-1bβ, H-1d, H-1f and H-1h), 4.500 (d, 0.6H,J_(1,2)2=7.8 Hz, H-1bα), 4.539, 4.543, 4.550 (d×3, 3H, J_(1,2)=8.4 Hz,J_(1,2)=8.5 Hz, J_(1,2)=8.4 Hz, H-1c or H-1e or H-1g), 4.702 (d, 0.4H,J_(1,2)=8.4 Hz, H-1aβ), 5.142 (d, 0.6H, H-1aα).

The spectrum used as the basis of the analysis is shown in FIG. 28.

125 MHz ¹³C-NMR δ; 24.84, 25.09, 25.35 (NHCOCH₃), 55.82 (C-2aα), 57.10,57.16 (C-2c, C-2e and C-2g), 58.44 (C-2aβ), 63.40, 63.57 (C-6a, C-6c andC-6e), 75.28, 75.34, 75.57 (C-2b, C-2d, C-2f and C-2h), 93.92 (C-1aα),97.61 (C-1aβ), 103.39, 103.45 (C-1c, C-1e and C-1g), 105.80, 105.85,105.95, 106.04 (C-1b or C-1d or C-1f or C-1h), 176.93, 177.40, 177.68,177.80, 178.27 (carbonyl).

The spectrum used as the basis of the analysis is shown in FIG. 29.

These results are not contradictory to the structure represented by theaforementioned formula (1) wherein n=3 (structure of sodiumβ-D-glucopyranosyluronate-[(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-(sodiumβ-D-glucopyranosyluronate)]₃-(1→3)-2-acetamido-2-deoxy-D-glucopyranose).

(6-4) Measurement Results of HA10

500 MHz ¹H-NMR δ; 2.001, 2.007, 2.016 (15H, NAc), 4.025 (dd, 0.6H,J_(1,2)=3.6 Hz, J_(2,3)=10.6 Hz, H-2aα), 4.441-4.465 (m, H-1bβ, H-1d,H-1f, H-1 h and H-1j), 4.499 (d, J_(1,2)=7.8 Hz, H-1bα), 4.538, 4.549(d×2, J_(1,2)=8.4 Hz, J_(1,2)=8.4 Hz, H-1c, H-1e, H-1g and H-1i), 4.701(d, J_(1,2)=8.4 Hz, H-1aβ), 5.141 (d, 0.6H, H-1aα).

The spectrum used as the basis of the analysis is shown in FIG. 30.

125 MHz ¹³C-NMR δ; 24.84, 25.09, 25.35 (NHCOCH₃), 55.82 (C-2aα), 57.10,57.16 (C-2c, C-2e, C-2g and C-2i), 58.44 (C-2aβ), 63.37, 63.57 (C-6a,C-6c, C-6e, C-6g and C-6i), 75.35, 75.58 (C-2b, C-2d, C-2f, C-2h andC-2j), 93.92 (C-1aα), 97.61 (C-1aβ), 103.39, 103.46 (C-1c, C-1e, C-1gand C-1i), 105.80, 105.86, 105.94, 106.05 (C-1b, C-1d, C-1f, C-1 h andC-1j), 176.94, 176.99, 177.41, 177.68, 177.81, 178.30 (carbonyl).

The spectrum used as the basis of the analysis is shown in FIG. 31.

These results are not contradictory to the structure represented by theaforementioned formula (1) wherein n=4 (structure of sodiumβ-D-glucopyranosyluronate-[(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-(sodiumβ-D-glucopyranosyluronate)]₄-(1→3)-2-acetamido-2-deoxy-D-glucopyranose).

(6-5) Measurement Results of HA12

500 MHz ¹H-NMR δ; 2.002, 2.006, 2.016 (18H, NAc), 4.025 (dd, 0.6H,J_(1,2)=3.6 Hz, J_(2,3)=10.7 Hz, H-2aα), 4.440-4.465 (m, H-1bβ, H-1d,H-1f, H-1 h, H-1j and H-1l), 4.498 (d, J_(1,2)=7.8 Hz, H-1bα), 4.538,4.549 (d×2, J_(1,2)=8.4 Hz, J_(1,2)=8.4 Hz, H-1c, H-1e, H-1g, H-1i andH-1k), 4.701 (d, J_(1,2)=8.5 Hz, H-1aβ), 5.141 (d, 0.6H, H-1aα).

The spectrum used as the basis of the analysis is shown in FIG. 32.

125 MHz ¹³C-NMR δ; 24.84, 25.09, 25.35 (NHCOCH₃), 55.82 (C-2aα), 57.09,57.16 (C-2c, C-2e, C-2g and C-2i), 58.44 (C-2aβ), 63.37 (C-6a, C-6c,C-6e, C-6g and C-6i), 75.35, 75.58 (C-2b, C-2d, C-2f, C-2h and C-2j),93.92 (C-1aα), 97.61 (C-1aβ), 103.40, 103.45 (C-1c, C-1e, C-1g andC-1i), 105.80, 105.86, 106.05 (C-1b, C-1d, C-1f, C-1 h and C-1j),176.95, 177.40, 177.80, 178.31 (carbonyl).

The spectrum used as the basis of the analysis is shown in FIG. 33.

These results are not contradictory to the structure represented by theaforementioned formula (1) wherein n=5 (structure of sodiumβ-D-glucopyranosyluronate-[(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-(sodiumβ-D-glucopyranosyluronate)]₅-(1→3)-2-acetamido-2-deoxy-D-glucopyranose).

(6-6) Measurement Results of HA14

500 MHz ¹H-NMR δ; 2.001, 2.006, 2.016 (21H, NAc), 4.024 (dd, 0.6H,J_(1,2)=3.6 Hz, J_(2,3)=10.6 Hz, H-2aα), 4.439-4.464 (m, H-1bβ, H-1d,H-1f, H-1 h, H-1j, H-1l and H-1n), 4.498 (d, J_(1,2)=7.8 Hz, H-1bα),4.537, 4.549 (d×2, J_(1,2)=8.4 Hz, J_(1,2)=8.3 Hz, H-1c, H-1e, H-1g,H-1i, H-1k and H-1m), 4.701 (d, J_(1,2)=8.4 Hz, H-1aβ), 5.141 (d, 0.6H,H-1aα).

The spectrum used as the basis of the analysis is shown in FIG. 34.

125 MHz ¹³C-NMR δ; 24.84, 25.09, 25.34 (NHCOCH₃), 55.81 (C-2aα), 57.09,57.15 (C-2c, C-2e, C-2g and C-2i), 58.43 (C-2aβ), 63.36, 63.57 (C-6a,C-6c, C-6e, C-6g and C-6i), 75.35, 75.58 (C-2b, C-2d, C-2f, C-2h andC-2j), 93.91 (C-1aα), 97.61 (C-1aβ), 103.40 (C-1c, C-1e, C-1g and C-1i),105.80, 105.85, 105.94, 106.07 (C-1b, C-1d, C-1f, C-1h and C-1j),176.95, 177.40, 177.67, 177.80, 178.30 (carbonyl).

The spectrum used as the basis of the analysis is shown in FIG. 35.

These results are not contradictory to the structure represented by theaforementioned formula (1) wherein n=6 (structure of sodiumβ-D-glucopyranosyluronate-[(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-(sodiumβ-D-glucopyranosyluronate)]₆-(1→3)-2-acetamido-2-deoxy-D-glucopyranose).

(6-7) Measurement Results of HA16

500 MHz ¹H-NMR δ; 2.002, 2.006, 2.016 (24H, NAc), 4.024 (dd, 0.6H,J_(1,2)=3.6 Hz, J_(2,3)=10.6 Hz, H-2aα), 4.434-4.464 (m, H-1bβ, H-1d,H-1f, H-1 h, H-1j, H-1l, H-1n and H-1p), 4.498 (d, J_(1,2)=7.8 Hz,H-1bα), 4.537, 4.549 (d×2, J_(1,2)=8.4 Hz, J_(1,2)=8.3 Hz, H-1c, H-1e,H-1g, H-1i, H-1k, H-1m and H-1o), 4.701 (d, J_(1,2)=8.4 Hz, H-1aβ),5.141 (d, 0.6H, H-1aα).

The spectrum used as the basis of the analysis is shown in FIG. 36.

125 MHz ¹³C-NMR δ; 24.84, 25.09, 25.35 (NHCOCH₃), 55.82 (C-2aα), 57.10,57.16 (C-2c, C-2e, C-2g, C-21, C-2k, C-2m and C-2o), 58.44 (C-2aβ),63.37, 63.57 (C-6a, C-6c, C-6e, C-6g, C-61, C-6k, C-6m and C-6o), 75.35,75.58 (C-2b, C-2d, C-2f, C-2h, C-2j, C-21, C-6n and C-6p), 93.92(C-1aα), 97.61 (C-1aβ), 103.40 (C-1c, C-1e, C-1g, C-1i, C-1k, C-1m andC-1o), 105.80, 105.86, 105.95, 106.08 (C-1b, C-1d, C-1f, C-1h, C-1j,C-1l, C-1n and C-1p), 176.98, 177.41, 177.68, 177.81, 178.33 (carbonyl).

The spectrum used as the basis of the analysis is shown in FIG. 37.

These results are not contradictory to the structure represented by theaforementioned formula (1) wherein n=7 (structure of sodiumβ-D-glucopyranosyluronate-[(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-(sodiumβ-D-glucopyranosyluronate)]₇-(1→3)-2-acetamido-2-deoxy-D-glucopyranose).

(6-8) Measurement Results of HA48

500 MHz ¹H-NMR δ; 1.998, 2.005, 2.016 (72H, NAc), 4.024 (dd, 0.6H,J_(1,2)=3.6 Hz, J_(2,3)=10.6 Hz, H-2aα), 4.395-4.470 (m, GlcA-unit),4.498 (d, J_(1,2)=8.0 Hz, H-1bα), 4.537, 4.610 (d×2, J_(1,2)=8.2 Hz,J_(1,2)=7.8 Hz, GlcNAc-unit), 4.700 (d, J_(1,2)=8.3 Hz, H-1aβ), 5.141(d, 0.6H, J_(1,2)=3.6 Hz, H-1aα).

The spectrum used as the basis of the analysis is shown in FIG. 38.

These results are not contradictory to the structure represented by theaforementioned formula (1) wherein n=23 (structure of sodiumβ-D-glucopyranosyluronate-[(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-(sodiumβ-D-glucopyranosyluronate)]₂₃-(1→3)-2-acetamido-2-deoxy-D-glucopyranose).

(6-9) Measurement Results of HA50

500 MHz ¹H-NMR δ; 2.005, 2.017 (75H, NAc), 4.024 (dd, 0.6H, J_(1,2)=3.6Hz, J_(2,3)=10.6 Hz, H-2aα), 4.390-4.470 (m, GlcA-unit), 4.498 (d,J_(1,2)=7.9 Hz, H-1bα), 4.537, 4.610 (d×2, J_(1,2)=8.1 Hz, J_(1,2)=8.1Hz, GlcNAc-unit), 4.701 (d, J_(1,2)=8.4 Hz, H-1aβ), 5.141 (d, 0.6H,J_(1,2)=3.6 Hz, H-1aα).

The spectrum used as the basis of the analysis is shown in FIG. 39.

These results are not contradictory to the structure represented by theaforementioned formula (1) wherein n=24 (structure of sodiumβ-D-glucopyranosyluronate-[(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-(sodiumβ-D-glucopyranosyluronate)]₂-(1→3)-2-acetamido-2-deoxy-D-glucopyranose).

(6-10) Measurement Results of HA52

500 MHz ¹H-NMR δ; 2.005, 2.017 (78H, NAc), 4.024 (dd, 0.6H, J_(1,2)=3.6Hz, J_(2,3)=10.6 Hz, H-2aα), 4.375-4.475 (m, GlcA-unit), 4.498 (d,J_(1,2)=8.2 Hz, H-1bα), 4.536, 4.610 (d×2, J_(1,2)=8.1 Hz, J_(1,2)=8.1Hz, GlcNAc-unit), 4.701 (d, J_(1,2)=8.4 Hz, H-1aβ), 5.141 (d, 0.6H,J_(1,2)=3.4 Hz, H-1aα).

The spectrum used as the basis of the analysis is shown in FIG. 40.

These results are not contradictory to the structure represented by theaforementioned formula (1) wherein n=25 (structure of sodiumβ-D-glucopyranosyluronate-[(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-(sodiumβ-D-glucopyranosyluronate)]₂₅-(1→3)-2-acetamido-2-deoxy-D-glucopyranose).

(7) Impurities Other than HA Oligosaccharides

Contents of the following impurities in the fractions were measured.

(7-1) Protein

Protein content was measured by using BioRad Protein Assay kit (producedby BioRad) and bovine serum albumin as a standard substance.

(7-2) DNA

DNA content was measured by the threshold method (DNA measuringapparatus: Threshold (Molecular Device, U.S.A.).

(7-3) Endotoxin

Endotoxin content was measured by the Limulus test method usingToxycolor System (trade name, produced by Seikagaku Corporation).

The measurement results for the protein, DNA and endotoxin contents inthe fractions are shown in Table 7.

TABLE 7 Sample Protein (%) DNA Endotoxin (pg/mL) HA4 Below Below 0.5detection detection limit limit HA6 Below Below 0.1 detection detectionlimit limit HA8 Below Below 0.1 detection detection limit limit HA10Below Below 0.7 detection detection limit limit HA12 Below Below 0.1detection detection limit limit HA14 Below Below 0.3 detection detectionlimit limit

From these results, it was concluded that the contents of protein andDNA in the fractions of the present invention were all below detectionlimit, the contents of endotoxin were all at a level that did notsubstantially influence, and thus they did not substantially containthem.

Example 2 Hsp expression promoting action <Materials Etc.> (1) TestSubstances Etc.

The test substances used in this example etc. are explained first.

Test substance (the abbreviations mentioned in the parentheses are usedin the following descriptions. In the following formulas, GlcA standsfor a glucuronic acid residue, GlcNAc for an N-acetylglucosamineresidue, Gal for a galactose residue, (6S) for 6-O-sulfate and — for aglycosidic linkage, and Δ indicates that the saccharide is anunsaturated saccharide.

Saturated HA disaccharide (HA2)

-   -   GlcAβ1-3GlcNAc

Unsaturated HA disaccharide (ΔHA2)

-   -   ΔGlcAβ1-3GlcNAc

Saturated HA tetrasaccharide (HA4)

-   -   GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc

Unsaturated HA tetrasaccharide (ΔHA4)

-   -   ΔGlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc

Saturated HA hexasaccharide (HA6)

-   -   GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc

Unsaturated HA hexasaccharide (ΔHA6)

-   -   ΔGlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc

Saturated HA octasaccharide (HA8)

-   -   GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc

Saturated HA decasaccharide (HA10)

-   -   GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAc

Saturated HA dodecasaccharide (HA12)

-   -   GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ1-4GlcAβ1-3GlcNAcβ-4GlcAβ1-3GlcNAcβ-4GlcAβ1-3GlcNAc

Mixture of Saturated HA disaccharide to octadecasaccharide (HA2-18)

Mixture of Saturated HA octadecasaccharide to tetradecasaccharide (HA8-14)

HA (weight average molecular weight: 840,000, HA84)

Gal(6S)β1-4GlcNAc(6S) (L4)

Gal(6S)β1-4GlcNAc(6S)β1-3Gal(6S)β1-4GlcNAc (6S) (L4L4)

As the saturated HA oligosaccharides, the HA oligosaccharide fractionsproduced in Preparation Example 1 mentioned above were used.

The unsaturated HA oligosaccharides were obtained by treating HA withchondroitin AC-1 lyase (Chondroitinase ACI Flavo, produced by SeikagakuCorporation) and fractionating the decomposition product in the samemanner as in Preparation Example 1 (refer to Preparation Example 2).

L4 and L4L4 were prepared by the method described in InternationalPatent Publication WO96/16973.

The test substances were dissolved in physiological saline to aconcentration predetermined according to the following pharmaceuticalefficacy tests. Endotoxin concentrations of the solutions inphysiological saline were all below 0.3 EU/mL, and iron contents of thesame were all 20 ppm or less.

(2) Cell and Medium K562 Cell (JCRB0019, Human Leukemia Cell) and PC-12Cell (JCRB0733)

K562 cells were cultured in RPMI 1640 medium containing 10% fetal bovineserum (FBS).

PC-12 cells were cultured in a medium obtained by adding 10 mL of 20%aqueous solution of glucose to 575 mL of DMEM medium (containingD-glucose (1,000 mg/l), L-glutamine (4 mM), sodium pyruvate (110 mg/l)and sodium bicarbonate (3.7 g/l)) containing 10% equine serum and 5%neonatal calf serum (FCS).

<1> Action on Phosphorylation of HSF1

It is known that HSF1 (heat shock factor 1) protein is a transcriptionfactor of Hsp70 (J. Biol. Chem., 271, pp. 3355-3358, 1996). Moreover,the HSF1 protein can be activated by phosphorylation, and thisphosphorylation can be detected based on band shift inSDS-polyacrylamide gel electrophoresis (SDS-PAGE) (apparent molecularweight shifts from about 66 kDa to about 81 kDa). By utilizing thisfact, the HA oligosaccharides of various sizes were allowed to act onthe K562 cells given with stress, and the aforementioned band shift wasdetected to investigate the degree of the phosphorylation (activation)of HSF1.

(1) Experiment 1

HA4, ΔHA4, HA6, HA8, HA2-18 and HA 8-14 were each added to a mediumcontaining the aforementioned cells at a concentration of 1, 10 or 100ng/mL. Then, the cells were immediately transferred into an environmentat 42° C. to be given with heat shock and incubated for 20 minutes.After the incubation, the cells were collected by centrifugation, andSDS-PAGE (gel concentration: 10%) was performed. Thereafter, Westernblotting was performed by using anti-HSF1 monoclonal antibodies(produced by Stressgene) as primary antibodies and goat anti-mouse IgGmonoclonal antibodies (produced by Jackson Lab) as secondary antibodiesto detect the shift of a band of 66 kDa (HSF1 not phosphorylated) to aband of 81 kDa (phosphorylated HSF1).

As a result, in the cells added with HA4, ΔHA4, HA6 or HA2-18, the shiftto the 81 kDa band was observed depending on the amount of the added HAoligosaccharide. Definite shift was not seen with HA8-14. Conversely,inhibition of the band shift was observed with HA8.

(2) Experiment 2

In order to confirm the reproducibility of Experiment 1, the band shiftwas detected in the same manner as in Experiment 1 by using HA4, ΔHA4,HA8 and HA84 except that HA was added in a single kind of amount of 100ng/mL and the heat shock was given at 43° C. The results are shown inFIG. 41. In FIG. 41, the first lane from the left indicates the resultobtained with an extract of cells incubated at 37° C. (no addition of HAoligosaccharide), the second lane indicates the result obtained with anextract of cells subjected to 43° C. (no addition of HAoligosaccharide), and the first lane from the right indicates the resultof detection of HSF1 (derived from bacteria) as a standard.

As seen from the result shown in FIG. 41, the band shift was observedfor the cells added with HA4 or ΔHA4, and the amounts of both HSF1 of 66kDa and 81 kDa per se were also increased. On the other hand, with HA8,increase of the amount of HSF1 of 66 kDa was observed, whereas the bandshift to the band of 81 kDa was not observed. Moreover, the shift wasnot seen with HA84.

(3) Experiment 3 (Comparative Experiment)

In order to investigate whether the action observed in Experiment 2 wasspecific to the saccharide chain basic structure of HA oligosaccharides,the following comparative experiment was performed by using chondroitinoligosaccharides. Chondroitin (referred to as Ch hereinafter) isdifferent from HA only in that GlcNAc in HA is replaced with agalactosamine residue (GalNAc), and they have common characteristicsthat they are commonly glycosaminoglycans, they do not have a sulfategroup etc. Therefore, it can be said that Ch is a substance extremelystructurally analogous to HA, and the same shall also apply to theoligosaccharides thereof.

Saturated Ch tetrasaccharide (Ch4)

-   -   GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc

Saturated Ch hexasaccharide (Ch6)

-   -   GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc

Saturated Ch octasaccharide (Ch8)

-   -   GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAcβ1-4GlcAβ1-3GalNAc

These saturated Ch oligosaccharides were obtained by treating Ch withdimethyl sulfoxide (DMSO) containing HCl and fractionating the obtaineddecomposition product for each size through anion exchangechromatography according to the method of Nagasawa et al. (Carbohyd.Res., 141, pp. 99-110, 1985).

The same experiment as Experiment 2 was performed by using theseoligosaccharides. As a result, the band shift of HSF1 or increase of theamount of HSF1 per se was not observed for any cells added with the Choligosaccharides.

By this experiment, it was demonstrated that the Ch oligosaccharides didnot have actions for activating HSF1 and promoting expression of HSF1,which were observed for the HA oligosaccharides. This indicates thatsuch actions should be specific to the saccharide chain basic structuresof HA oligosaccharides.

By these experiments, it was revealed that the HA tetrasaccharide and afraction containing it (HA2-18) particularly notably activated HSF1.

<2> Action on Expression of Hsp

Since it was revealed from <1> that the HA tetrasaccharide more notablyactivated the factor required for the expression of Hsp70 (HSF1)compared with HA of other sizes, the following experiment was performedin order to investigate whether the expression of Hsp in K562 cellsgiven with stress should be actually increased more notably by theaction of HA tetrasaccharide compared with cases utilizing HA of othersizes.

HA2, ΔHA4, HA6, HA84 and L4L4 were each added to a medium containing theaforementioned cells (37° C.) at a concentration of 1, 10 or 100 ng/mL.Then, the cells were immediately transferred into an environment at 43°C. to be given with heat shock and incubated for 20 minutes. Then, thecells were returned to 37° C., then further incubated for 2 hours andcollected by centrifugation, and SDS-PAGE was performed in the samemanner as in <1> mentioned above. Thereafter, Western blotting wasperformed by using anti-Hsp72 monoclonal antibodies (produced byAmersham) as primary antibodies and goat anti-rabbit IgG monoclonalantibodies (produced by Jackson Lab) as secondary antibodies to detectHsp72. Hsp72 is one of the members of Hsp70 family and is the mosttypical one, and it is known that its expression is induced and promotedby stress.

The results are shown in FIG. 42. The leftmost lane in FIG. 42 aindicates the results of the detection of Hsp72 in a similar manner withgiving no heat shock (37° C.) and addition of no test substance, and therightmost lane indicates the results of detection of standard Hsp72(Hsp72 derived from bacteria, bHsp72) in a similar manner. FIG. 42 bshows the results of detection of the expression of Hsp72 in a similarmanner with addition of ΔHA4 to the cells that were not given with heatshock.

As clearly seen from the results shown in FIG. 42 a, when a heat shock(43° C.) was given (the second lane from the left), the expression ofHsp was enhanced compared with the case where a test substance was notadded at 37° C. (the leftmost lane). Further, in the presence of each ofthe HA oligosaccharides, the expression of Hsp was further enhanced, andthe expression of Hsp was particularly strongly enhanced in the presenceof ΔHA4 (the third to fifth lanes from the left).

Thus, strong expression of Hsp72 was observed in the cells added withΔHA4 (given with the heat shock), whereas such strong expression ofHsp72 as in the cells added with ΔHA4 was not observed in the cell addedwith HA of other sizes.

Moreover, in the cells that were not given with the heat shock,enhancement of the expression of Hsp72 by ΔHA4 was not observed. Fromthis fact, it was revealed that, although the HA tetrasaccharidescarcely affect the expression of Hsp under no stress condition, itextremely quickly and notably enhance the expression of Hsp if stress isonce loaded.

<3> Cell Death Inhibitory Action

Since it was revealed that the HA tetrasaccharide more strongly enhancedthe expression of Hsp in the cells under stress compared with HAoligosaccharides of other sizes, the following experiment was performedin order to investigate whether the HA oligosaccharides actuallysuppressed death of cells subjected to stress more strongly or not.

It is known that the PC-12 cells suffer from apoptosis in a culturebroth that does not contain blood serum.

Therefore, each of ΔHA2, HA4, ΔHA4, HA6, ΔHA6, HA8, HA10, HA12, HA84, L4and L4L4 was added to a culture broth containing PC-12 cells at aconcentration of 100 ng/mL (containing no blood serum), 24 hours after,the cells were stained with trypan blue, and a ratio of the number ofviable cells (cells not stained with trypan blue) to the total cellnumber was calculated.

The results are shown in FIG. 43. As seen from the results shown in FIG.43, the HA tetrasaccharides, especially HA4, more notably suppressed thecell death compared with HA oligosaccharides of other sizes or theoligosaccharides of other kinds.

<4> Cell and Tissue Protection Action 1. Cell and Tissue ProtectionAction in Organ Preservation Aspect (1) Preparation of OrganPreservation Solution

A Euro-Collins solution (Am. J. Surg., 179, pp. 154-160, 2000)containing 100 ng/mL of HA tetrasaccharide was used as a test solution(also referred to as “HA4(+)” hereafter). As a control, the Euro-Collinssolution (also referred to as “HA4(−)” hereafter) was used.

(2) Administration of Test Substance Etc.

SD male rats of 11-week old were divided into a “HA4(+) used” group(n=5), “HA4(−) used” group (n=5) and normal group (n=5). After abdominalsection, organs (livers) were extracted. The organs of the “HA4(+) used”group and “HA4(−) used” group were perfused with 40 mL of each solutionuntil the color of the whole livers became light due to bleeding andthen incubated at 37° C. for 2 hours.

(3) Evaluation

After the incubation, for each group, a ratio of wet weight/dry weightof organ (parameter of degree of edema caused by tissue affection) wascalculated, a HE-stained tissue section was observed by using an opticalmicroscope (visual inspection of tissue affection), and image analysisof chromatin concentration and analysis by the TUNEL method (terminaldeoxynucleotidyl transferase (TdT)-mediated nick end labeling method, J.Cell. Biol., 119, pp. 493-501, 1992, evaluation of degree of cell death)were performed.

(3-1)

The results for the ratio of wet weight/dry weight of organ are shown inFIG. 44. The symbol of * in FIG. 44 indicates the presence ofsignificant difference with p<0.05 (Williams multiple range test).

As seen from the results shown in FIG. 44, the ratio of wet weight/dryweight of organ was significantly decreased in the “HA4(+)” used groupcompared with the “HA4(−)” used group. This indicated that the HAtetrasaccharide significantly suppressed edema due to tissue affectioncaused by preservation of organ.

(3-2)

As a result of the observation of HE-stained tissue section by using anoptical microscope, edema, histolysis (disappearance of plasma),pyknosis etc. were observed in the “HA4(−)” used group. On the otherhand, images substantially similar to those of the normal group wereobserved for the “HA4(+)” used group, and remarkable tissue affection,which was observed for the “HA4(−)” used group, was not observed.

(3-3) Image Analysis for Chromatin Concentration

As one method for evaluating degree of cell death, image analysis ofcellular chromatin concentration was performed (Image-Pro PLUS™ Version3.0.1, produced by Media Cybernetics) by using HE-stained tissuesections to compare image densities. If cell death occurs and chromatinis concentrated, that portion will be deeply stained with HE. If thereare more cells in which chromatin is concentrated, density of the wholemicroscopic image is increased. Therefore, the evaluation was performedby using the density as an index.

The results are shown in FIG. 45. The symbol of * in FIG. 45 indicatesthe presence of a significant difference with p<0.05 (Williams multiplerange test).

As seen from the results shown in FIG. 45, the image density wassignificantly increased in the “HA4(−)” used group compared with thenormal group, whereas significant increase of the image density was notobserved for the “HA4(+)” used group compared with the normal group.

(3-4) Analysis by TUNEL Method

As one method for evaluating degree of cell death, degree offragmentation of DNA was analyzed by using the TUNEL method. It is knownthat, if a cell causes apoptosis, DNA is fragmented. The TUNEL method isa method for staining termini of DNA, and if there are many cells inwhich DNA is fragmented, degree of TUNEL staining is increased.Therefore, it can be used as an index of the degree of cell death(apoptosis).

Tissue sections after storage were subjected to the TUNEL staining andvisually inspected by using an optical microscope to count the number ofcells stained by the TUNEL method per 1 mm². At the same time, thedegree of tissue affection of the tissue sections was evaluated withfour grades including “severe”, “moderate”, “slight” and “extremelyslight”. In addition, this evaluation was performed as a blind test inorder to secure objectivity. The results of the former are shown in FIG.46, and the results of the latter are shown in FIG. 47. The symbol of *in FIG. 46 indicates the presence of significant difference with p<0.05(Williams multiple range test).

As seen from the results shown in FIG. 46, an extremely large number ofTUNEL-stained cells were observed for the “HA4(−)” used group, whereasTUNEL-stained cells observed for the “HA4(+)” used group were extremelyfew and the number was at a level similar to that of the normal group.Further, as seen from the results shown in FIG. 47, “severe” or“moderate” tissue affection was observed in 80% of the tissue sectionsin the “HA4(−)” used group, whereas “severe” tissue affection was notobserved and “moderate” tissue affection was observed only in 20% of thetissue sections in the “HA4(+)” used group.

Based on the results mentioned above, it was revealed that the HAoligosaccharide (HA tetrasaccharide) had superior cell and tissueprotection effect in the organ preservation aspect.

2. Cell and Tissue Protection Action in Aspect of Effect on Ulcer (1)Preparation of Test Solutions for Administration

A test substance was dissolved in carboxymethylcellulose (CMC) to obtaina test solution for administration. The concentration was determineddepending on the dose mentioned below so that the volume of the solutionto be administered should become 10 mL/kg.

Moreover, as a positive control solution, a conventional agent fortreating gastritis and gastric ulcer (teprenone, geranylgeranylacetone,trade name: Selbex, Eisai) dissolved in CMC was used. The concentrationwas determined so that the dose and the volume of the solution to beadministered should become 1 mg/kg (clinical dose) and 10 mL/kg,respectively. As a negative control solution, 0.5% CMC was used.

(2) Administration of Test Substance Etc.

SD male rats of 5-week old were divided into a “negative controlsolution” administered group (n=8), “HA4 4 mg/kg” administered group(n=8), “HA4 20 mg/kg” administered group (n=8), “HA4 100 mg/kg”administered group (n=8) and “positive control solution” administeredgroup (n=8).

The rats of the groups were starved from 16:00 of the day before thefirst administration day, orally administered with each of theaforementioned solutions at 8:00 of the next day and arrested by waterimmersion. The arrest by water immersion was continued until 8:00 of thenext day (24 hours). In the course of the arrest, each solution wasadministered in the same manner as the first administration at 16:00 and24:00.

Then, the rats were dissected, and the stomachs were extracted. Theextracted stomachs were expanded by injection of 10% buffered formalinto smoothen the holds and fixed. Then, the blood adhering to walls ofstomachs was removed by washing with water.

(3) Evaluation

The area ratio of ulcerous portion degenerated in a dark brown color(ulcer area/glandular stomach area) was measured by using an imageanalysis apparatus (Image-Pro PLUS™ Version 3.0.1, produced by MediaCybernetics). The results are shown in FIG. 48. The symbols * and ** inFIG. 48 indicate significant differences with p<0.05 and p<0.01,respectively (Williams multiple range test).

As seen from the results shown in FIG. 48, the area ratio of ulcerousportion was significantly reduced in all the HA4 administered groupscompared with the negative control solution administered group. Inparticular, the HA4 20 mg/kg administered group and 100 mg/kgadministered group showed an area ratio of ulcerous portion lower thanthat observed for the positive control solution group.

The results mentioned above revealed that the HA oligosaccharide (HAtetrasaccharide) had superior cell and tissue protection effect also inthe aspect of the effect on ulcer. It was also revealed that theaforementioned superior effect could be obtained even by oraladministration of the HA oligosaccharide. Furthermore, since the ratswere administered with the HA oligosaccharide (HA tetrasaccharide)beforehand and then arrested by water immersion in this experiment, andit takes a certain period of time from the start of the arrest by waterimmersion until ulcer is generated, it can be also said that the resultsof this experiment suggests that the HA oligosaccharide (HAtetrasaccharide) has a prophylactic effect.

3. Cell and Tissue Protection Action in Aspect of Effect on HepatopathyExperiment 1: Experiment Using Carbon Tetrachloride Hepatopathy Model(1) Preparation of Test Solutions for Administration

A test substance was dissolved in physiological saline to obtain a testsolution for administration. The concentration was determined dependingon the dose mentioned below so that the volume of the solution to beadministered should become 5 mL/kg.

Moreover, as a positive control solution, FAD (flavine adeninedinucleotide, Wakamoto Pharmaceutical) of which effect on the carbontetrachloride hepatopathy model had been reported (Yakubutsu Ryouhou(Pharmacotherapy), vol. 9, Special edition, pp. 46-53), was used at adose of 10 mg/kg. Further, physiological saline was used as a negativecontrol solution.

(2) Administration of Test Substance Etc.

SD male rats of 5-week old were divided into a “negative controlsolution” administered group (n=8), “HA4 2 mg/kg” administered group(n=8), “HA4 10 mg/kg” administered group (n=8), “HA4 50 mg/kg”administered group (n=8), “positive control solution” administered group(n=8) and “normal” group (n=8).

The rats of the groups were starved from 16:00 of the day before thefirst administration day, orally administered with 100 mg/kg of carbontetrachloride (CCl₄) before 8:00 of the next day and thenintraperitoneally administered with each of the aforementioned solutionsat 8:00. Thereafter, the rats were bred with maintaining starvation, andadministered with each of the aforementioned solutions in the samemanner as the first administration at 16:00 and 24:00. The rats were fedafter the administration at 24:00 and dissected at 8:00 on the next day.

(3) Evaluation

After the dissection, blood was collected, and GOT activity andleucocyte count were measured. The GOT activity was measured by using aclinical chemistry autoanalyzer (COBAS MIRA S, Nippon Roche), and theleucocyte count was measured by using an automated hematology analyzer(Sysmex K-2000, produced by Sysmex).

The results of the former are shown in FIG. 49, and the results of thelatter are shown in FIG. 50. The symbol of * in FIGS. 49 and 50indicates presence of significant difference with p<0.05 (Williamsmultiple range test).

As seen from the results shown in FIG. 49, significant reduction of theGOT activity was observed in the HA4 50 mg/mL administered groupcompared with the negative control solution administered group.Moreover, as seen from the results shown in FIG. 50, significantdecrease of the peripheral leucocyte count was observed for all the HA4administered groups compared with the negative control solutionadministered group, and the leucocyte count was decreased compared evenwith FAD. These results revealed that the HA tetrasaccharide had a celland tissue protection effect also in the aspect of the effect onhepatopathy. Furthermore, since the rats were administered with carbontetrachloride beforehand and then administered with the HAoligosaccharide (HA tetrasaccharide) in this experiment, and it takes acertain period of time from the administration of carbon tetrachlorideuntil hepatopathy is generated by carbon tetrachloride, it can be alsosaid that the results of this experiments suggests that the HAoligosaccharide (HA tetrasaccharide) has a prophylactic effect.

Moreover, in this experiment, IL-10 and IL-8 concentrations inperipheral blood were also measured.

It has been reported that IL-10 is associated with one of the stressproteins (proteins that are expressed when a cell receives stress andhave an action for protecting the cell), Hsp70 (J. Immunol., 164 (5),pp. 2711-2717, 2000), and it has also been reported that IL-10suppressed hepatitis caused by concanavalin A (Autoimmunity, 31 (2), pp.75-83, 1999).

Moreover, it has been revealed that the expression amount of IL-8 wasincreased in a hepatopathy model using cadmium or alcohol (Toxicologyand Applied Pharmacology, 163, pp. 231-9, 2000; Acta Gastro-EnterologicaBelgica, 57 (3-4), pp. 255-9, 1994), and it has also been reported thatthe expression of IL-8 was further inhibited by induction of Hsp70expression (Journal of Immunology, 164, pp. 5416-23, 2000).

IL-10 in peripheral blood was measured by using IL-10 Rat ELISA(produced by ENDOGEN). IL-8 was measured by using Panatest A Series RatCINC-1 (IL-8) (Panapharm Laboratories). The measurement results forIL-10 are shown in FIG. 51, and the measurement results for IL-8 areshown in FIG. 52. The symbol of ** in FIGS. 51 and 52 indicates presenceof significant difference with p<0.01 (Williams multiple range test).

From the results shown in FIG. 51, it can be seen that the blood IL-10level was increased in a manner dependent on the dose of HA4, and theblood IL-10 level was significantly higher in the HA4 50 mg/kgadministered group compared with the negative control solutionadministered group. Further, from the results shown in FIG. 52, it canbe seen that the blood IL-8 level was decreased in a manner dependent onthe dose of HA4.

These results revealed that the HA oligosaccharide (HA tetrasaccharide)also had an IL-10 production promoting effect and IL-8 productioninhibitory effect. This also suggests a possibility that the HAoligosaccharide (HA tetrasaccharide) may exert the aforementioned celland tissue protection effect through promotion of the IL-10 productionor inhibition of the IL-8 production. Moreover, it was also demonstratedthat the HA oligosaccharide (HA tetrasaccharide) can be used also as anagent for treatment of diseases caused by decrease of IL-10 or increaseof IL-8 and diseases for which promotion of the IL-10 production orinhibition of the IL-8 production is desired.

Experiment 2: Experiment Using Concanavalin A Model

In order to confirm whether the HA oligosaccharide (HA tetrasaccharide)might be effective also on a hepatopathy model other than the carbontetrachloride hepatopathy model, an experiment was performed by using aconcanavalin A hepatopathy model.

(1) Preparation of Test Solution for Administration

The preparation, concentration, dose, volume to be administered etc. ofthe test solution are the same as those used in Experiment 1.

As a positive control solution, Stronger Neo-Minophagen C (MinophagenPharmaceutical) was used at a dose of 5 mg/kg. This dose is the maximumclinical dose of the drug. Moreover, physiological saline was used as anegative control solution.

(2) Administration of Test Substance Etc.

Balb/c mice of 8-week old were divided into a “negative controlsolution” administered group (n=12), “HA4 2 mg/kg” administered group(n=12), “HA4 10 mg/kg” administered group (n=12), “HA4 50 mg/kg”administered group (n=12), “positive control solution” administeredgroup (n=12) and “normal” group (n=8).

Concanavalin A (ConA, Sigma) was injected via the caudal vein at a doseof 15 mg/kg. Then, each of the aforementioned solutions was administeredvia the caudal vein. Thereafter, the mice were bred for 24 hours.

(3) Evaluation

The whole blood was collected from the abdominal aorta, and the GPTactivity and GOT activity were measured by a clinical chemistryautoanalyzer (COBAS MIRA S, Nippon Roche). Further, the liver wasextracted, and appearance of the liver was evaluated by visualinspection.

As a result, the average of GPT was about 5600 (I.U./L) in the “negativecontrol solution” administered group. On the other hand, it was about2200 (I.U./L) and about 1200 (I.U./L) in the “HA4 10 mg/kg” administeredgroup and the “HA4 50 mg/kg” administered group, respectively, and bothof the values were significantly lower than that observed for the“negative control solution” administered group (p<0.01, Dunnett multiplecomparison test). In addition, it was about 4000 (I.U./L) for the“positive control solution” administered group.

Further, the average of GOT was about 6000 (I.U./L) for the “negativecontrol solution” administered group. On the other hand, it was about2000 (I.U./L) and about 1000 (I.U./L) for the “HA4 10 mg/kg”administered group and the “HA4 50 mg/kg” administered group,respectively, and thus both of the values were significantly lower thanthat observed for the “negative control solution” administered group(p<0.01, Dunnett multiple comparison test). In addition, it was about4400 (I.U./L) for the “positive control solution” administered group.

Moreover, as a result of the visual inspection of the livers, both ofthe “HA4 10 mg/kg” administered group and the “HA4 50 mg/kg”administered group showed a lower degree of hepathopathy compared withthe “negative control solution” administered group and the “positivecontrol solution” administered group.

These results demonstrated that the HA oligosaccharide (HAtetrasaccharide) exhibited an effect also on hepatopathy caused byconcanavalin A, and it was suggested that the HA oligosaccharide (HAtetrasaccharide) was effective to various hepatopathies. Further, it canbe said that the results of this experiment suggest that the HAoligosaccharide (HA tetrasaccharide) has a prophylactic effect, like theresults obtained by using the carbon tetrachloride model.

In addition, safety of the agents of the present invention can beestimated from the high safety of the HA oligosaccharides themselves aswell as the results of the aforementioned examples.

INDUSTRIAL APPLICABILITY

The oligosaccharides of the present invention are useful as activeingredients of the drugs of the present invention and so forth.

The fractions of the present invention are HA oligosaccharide fractionsthat contain an HA oligosaccharide of a desired size and do notsubstantially contain oligosaccharides of other sizes and impurities,and are extremely useful as reagents for search of physiologicalactivities of HA oligosaccharides, standards for analyses, drugsthemselves or materials thereof.

Since an HA oligosaccharide of a specific size exhibits a remarkableeffect that cannot be observed for HA oligosaccharides of other sizes asclearly seen from the results of the aforementioned examples, the drugscontaining the oligosaccharides of the present invention as an activeingredient, especially Hsp expression promoter, are extremely useful.Further, if an HA oligosaccharide of such a specific size is selected,the dose can be reduced compared with HA oligosaccharides of other sizeswhile maintaining the same degree of effect as the HA oligosaccharidesof other sizes. Therefore, they are extremely advantageous also inaspect that they enable production of more inexpensive drugs havinghigher safety.

Furthermore, the cell death inhibitor, cell injury inhibitor and celland tissue protecting agent (e.g., organ preservation agent, antiulceragent, antihepatopathic agent, IL-10 production promoter or IL-8production suppressor) containing the oligosaccharides of the presentinvention as active ingredients are extremely useful, because theyexhibit superior pharmaceutical effects as clearly seen from the resultsof the aforementioned examples and exhibit high safety.

1. A method for promoting expression of a heat shock protein, or forinhibiting cell injury or cell death, or for treating a disease forwhich cell or tissue protection is desired, or for promoting productionof IL-10, or for inhibiting production of IL-8, which comprisesadministering an effective amount of a fraction comprising a hyaluronicacid tetrasaccharide comprising four saccharide residues and having thephysicochemical properties defined in the following (1) to (6): (1) whenthe fraction is analyzed by the detection methods mentioned in thefollowing (a) and (b) using gel filtration chromatography, the fractionshows a substantially single peak in both of the methods, and the peakshave peak areas defined in the following (a) and (b): (a) when thedetection is performed based on absorbance at 210 nm, a relative ratioof peak area of the tetrasaccharide to the sum of peak areas of total HAoligosaccharides in the fraction is 85% or more; (b) when the detectionis performed by using a differential refractometer, a relative ratio ofpeak area of the tetrasaccharide to the sum of peak areas of total HAoligosaccharides in the fraction is 98% or more; (2) when the fractionis analyzed based on absorbance at 210 nm by anion exchangechromatography, the fraction shows a substantially single peak, and arelative ratio of peak area of the tetrasaccharide to the sum of peakareas of total HA oligosaccharides in the fraction is 90% or more; (3)when the tetrasaccharide in the fraction is labeled with fluorescenceand then analyzed by electrophoresis, a single band is detected, andbands of HA oligosaccharides of other sizes are not detected; (4) when atheoretical value of monoisotopic molecular weight or average molecularweight of the tetrasaccharide constituting the fraction is taken as 1,an actual value of the same measured for the fraction by massspectrometry is 0.997 to 1.003 (relative value); (5) respectivedifferences between theoretical values (weight %) of carbon (C),hydrogen (H) and nitrogen (N) contents in the tetrasaccharideconstituting the fraction and values of the same actually measured forthe elements by elemental analysis of the fraction (weight %) are all inthe range of ±1 (weight %); and (6) the fraction does not substantiallycontain proteins, DNA and endotoxins to a subject in need thereof. 2.The method according to claim 1, wherein said method is a method forpromoting expression of a heat shock protein and wherein said subject inneed thereof is a subject in need of such promotion.
 3. The methodaccording to claim 1, wherein said method is a method for inhibitingcell injury or cell death, and wherein said subject in need thereof is asubject in need of such inhibition.
 4. The method according to claim 1,wherein said method is a method for treating a disease for which cell ortissue protection is desired, and wherein said subject in need thereofis a subject in need of such a treatment.
 5. The method according toclaim 4, wherein the disease is ulcer or hepatopathy.
 6. The methodaccording to claim 1, wherein said method is a method for promotingproduction of IL-10, and wherein said subject in need thereof is asubject in need of such promotion.
 7. The method according to claim 1,wherein said method is a method for inhibiting production of IL-8, andwherein said subject in need thereof is a subject in need of suchinhibition.
 8. The method according to claim 1, wherein the hyaluronicacid tetrasaccharide is a saturated hyaluronic acid tetrasaccharide. 9.A method for promoting expression of a heat shock protein, or forinhibiting cell injury or cell death, or for treating a disease forwhich cell or tissue protection is desired, or for promoting productionof IL-10, or for inhibiting production of IL-8, which comprisesadministering an effective amount of an isolated and substantially puretetrasaccharide of formula (1):

wherein n is 1, m is a proton or monovalent cation, and Ac represents anacetyl group to a subject in need thereof.
 10. The method according toclaim 9, wherein said method is a method for promoting expression of aheat shock protein and wherein said subject in need thereof is a subjectin need of such promotion.
 11. The method according to claim 9, whereinsaid method is a method for inhibiting cell injury or cell death, andwherein said subject in need thereof is a subject in need of suchinhibition.
 12. The method according to claim 9, wherein said method isa method for treating a disease for which cell or tissue protection isdesired, and wherein said subject in need thereof is a subject in needof such a treatment.
 13. The method according to claim 12, wherein thedisease is ulcer or hepatopathy.
 14. The method according to claim 1,wherein said method is a method for promoting production of IL-10, andwherein said subject in need thereof is a subject in need of suchpromotion.
 15. The method according to claim 1, wherein said method is amethod for inhibiting production of IL-8, and wherein said subject inneed thereof is a subject in need of such inhibition.